Group b streptococcus vaccine

ABSTRACT

This application relates to improved Group B Streptococcus (“GBS”) saccharide-based vaccines comprising combinations of GBS polysaccharides with polypeptide antigens, and vice versa, such that the polypeptide and the saccharide each contribute to the immunological response in a recipient. The combination is particularly advantageous where the saccharide and polypeptide are from different GBS serotypes. The combined antigens may be present as a simple combination where separate saccharide and polypeptide antigens are administered together, or they may be present as a conjugated combination, where the saccharide and polypeptide antigens are covalently linked to each other. Preferably, the immunogenic compositions of the invention comprise a GBS saccharide antigen and at least two GBS polypeptide antigens, wherein said GBS saccharide antigen comprises a saccharide selected form GBS serotype Ia, Ib, and III, and wherein said GBS polypeptide antigens comprise a combination of at least two polypeptide or fragments thereof selected from the antigen group consisting of GBS 80, GBS 91, GBS 104, GBS 147, GBS 173, GBS 276, GBS 305, GBS 313, GBS 322, GBS 328, GBS 330, GBS 338, GBS 358, GBS 361, GBS 404, GBS 656, GBS 690, and GBS 691.

This application is a divisional application of Ser. No. 10/527,672 filed on Mar. 11, 2005 as a national phase of PCT/US2003/029167 filed on Sep. 15, 2003, which claims the benefit of Ser. No. 60/410,839 filed on Sep. 13, 2002. These applications are incorporated herein by reference in their entireties.

This application incorporates by reference the contents of a 124 kb text file created on Sep. 6, 2013 and named “sequencelisting.txt,” which is the sequence listing for this application.

TECHNICAL FIELD

This invention relates to polysaccharides from the bacteria Streptococcus agalactiae (GBS) and to their use in immunisation.

BACKGROUND ART

Once thought to infect only cows, the Gram-positive bacterium Streptococcus agalactiae (or “group B streptococcus”, abbreviated to “GBS” (Ref 1) is now known to cause serious disease, bacteremia and meningitis, in immunocompromised individuals and in neonates. There are two types of neonatal infection. The first (early onset, usually within 5 days of birth) is manifested by bacteremia and pneumonia. It is contracted vertically as a baby passes through the birth canal. GBS colonises the vagina of about 25% of young women, and approximately 1% of infants born via a vaginal birth to colonised mothers will become infected. Mortality is between 50-70%. The second s a meningitis that occurs 10 to 60 days after birth. If pregnant women are vaccinated with type III capsule so that the infants are passively immunised, the incidence of the late onset meningitis is reduced but is not entirely eliminated.

The “B” in “GBS” refers to the Lancefield classification, which is based on the antigenicity of a carbohydrate which is soluble in dilute acid and called the C carbohydrate. Lancefield identified 13 types of C carbohydrate, designated A to O, that could be seriologically differentiated. The organisms that most commonly infect humans are found in groups A, B, D, and G. Within group B, strains can be divided into at least 9 serotypes (Ia, Ib, laic, II, III, N, V, VI, VII and VIII) based on the structure of their polysaccharide capsule. In the past, serotypes Ia, Ib, II, and III were equally prevalent in normal vaginal carriage and early onset sepsis in newborns. Type V GBS has emerged as an important cause of GBS infection in the USA, however, and strains of types VI and VIII have become prevalent among Japanese women.

The genome sequence of a serotype V strain 2603 VIR has been published (Ref. 2) and various polypeptides for use a vaccine antigens have been identified (Ref. 3). The vaccines currently in clinical trials, however, are based on polysaccharide antigens. These suffer from serotype-specificity and poor immunogenicity, and so there is a need for effective vaccines against S. agalactiae infection.

It is an object of the invention to provide further and improved GBS vaccines.

DISCLOSURE OF THE INVENTION

The inventors have realised that saccharide-based vaccines can be improved by using them in combination with polypeptide antigens, and vice versa, such that the polypeptide and the saccharide each contribute to the immunological response in a recipient. The combination is particularly advantageous where the saccharide and polypeptide are from different GBS serotypes.

The combined antigens may be present as a simple combination where separate saccharide and polypeptide antigens are administered together, or they may be present as a conjugated combination, where the saccharide and polypeptide antigens are covalently linked to each other.

Thus the invention provides an immunogenic composition comprising (i) one or more GBS polypeptide antigens and (ii) one or more GBS saccharide antigens. The polypeptide and the polysaccharide may advantageously be covalently linked to each other to form a conjugate.

Between them, the combined polypeptide and saccharide antigens preferably cover two or more GBS serotypes (e.g. 2, 3, 4, 5, 6, 7, 8 or more serotypes). The serotypes of the polypeptide and saccharide antigens may or may not overlap. For example, the polypeptide might protect against serogroup II or V, while the saccharide protects against either serogroups Ia, Ib, or III. Preferred combinations protect against the following groups of serotypes: (1) serotypes Ia and Ib, (2) serotypes Ia and II, (3) serotypes Ia and III, (4) serotypes Ia and IV, (5) serotypes Ia and V, (6) serotypes Ia and VI, (7) serotypes Ia and VII, (8) serotypes Ia and VIII, (9) serotypes Ib and II, (10) serotypes Ib and III, (11) serotypes Ib and IV, (12) serotypes Ib and V, (13) serotypes Ib and VI, (14) serotypes Ib and VII, (15) serotypes Ib and VIII, 16) serotypes II and III, (17) serotypes II and IV, (18) serotypes II and V, (19) serotypes II and VI, (20) serotypes II and VII, (21) serotypes II and VII, (22) serotypes III and IV, (23) serotypes III and V, (24) serotypes III and VI, (25) serotypes III and VII, (26) serotypes III and VIII, (27) serotypes IV and V, (28) serotypes IV and VI, (29) serotypes IV and VII, (30) serotypes IV and VIII, (31) serotypes V and VI, (32) serotypes V and VII, (33) serotypes V and VIII, (34) serotypes VI and VII, (35) serotypes VI and VIII, and (36) serotypes VII and VIII.

Still more preferably, the combinations protect against the following groups of serotypes: (1) serotypes Ia and II, (2) serotypes Ia and V, (3) serotypes Ib and II, (4) serotypes Ib and V, (5) serotypes III and II, and (6) serotypes III and V. Most preferably, the combinations protect against serotypes III and V.

Protection against serotypes II and V is preferably provided by polypeptide antigens. Protection against serotypes Ia, Ib and/or III may be polypeptide or saccharide antigens.

Preferably, the immunogenic composition comprises one or more serogroup V antigens or fragments thereof selected from the antigen group consisting of GBS 80, GBS 91, GBS 104, GBS 147, GBS 173, GBS 276, GBS 305, GBS 313, GBS 322, GBS 328, GBS 330, GBS 338, GBS 358, GBS 361, GBS 404, GBS 656, GBS 690, and GBS 691. Preferably, the composition comprises a composition of at least two of these GBS antigens or a fragment thereof.

In one embodiment, the immunogenic composition comprises a GBS saccharide antigen and at least two GBS polypeptide antigens or fragments thereof, wherein said GBS saccharide antigen comprises a saccharide selected from GBS serotype Ia, Ib, and III, and wherein said GBS polypeptide antigens comprise a combination of at least two polypeptide or a fragment thereof selected from the antigen group consisting of GBS 80, GBS 91, GBS 104, GBS 147, GBS 173, GBS 276, GBS 305, GBS 313, GBS 322, GBS 328, GBS 330, GBS 338, GBS 358, GBS 361, GBS 404, GBS 656, GBS 690, and GBS 691.

Preferably, the combination comprises GBS 80 or a fragment thereof. In one embodiment, the GBS polypeptide antigens comprise a combination of two GBS antigens or fragments thereof selected from the antigen group consisting of (1) GBS 80 and GBS 91, (2) GBS 80 and GBS 104, (3) GBS 80 and GBS 147, (4) GBS 80 and GBS 173, (5) GBS 80 and GBS 276, (6) GBS 80 and GBS 305, (7) GBS 80 and GBS 313, (8) GBS 80 and GBS 322, (9) GBS 80 and GBS 328, (10) GBS 80 and GBS 330, (11) GBS 80 and GBS 338, (12) GBS 80 and GBS 358, (13) GBS 80 and GBS 361, (14) GBS 80 and GBS 404, (14) GBS 80 and GBS 404, (15) GBS 80 and GBS 656, (16) GBS 80 and GBS 690, and (17) GBS 80 and GBS 691.

Still more preferably, the combination is selected from the antigen group consisting of (1) GBS 80 and GBS 338; (2) GBS 80 and GBS 361, (3) GBS 80 and GBS 305, (4) GBS 80 and GBS 328, (5) GBS 80 and GBS 690, (6) GBS 80 and GBS 691 and (7) GBS 80 and GBS 147. Even more preferably, the combination comprises GBS 80 and GBS 691.

In one embodiment, the composition comprises a combination at least three GBS polypeptide antigens. Preferably, this combination comprises GBS 80 and GBS 691.

Preferably, the immunogenic composition further comprises a GBS polypeptide or a fragment thereof of serogroup

The Polypeptide Antigen

The polypeptide is preferably: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of the even-numbered SEQ IDs 2-10966 from Ref. 3; (b) a polypeptide comprising an amino acid sequence having sequence identity to an amino acid sequence from in (a); or (c) a polypeptide comprising a fragment of an amino acid sequence from (a).

Within (a), preferred SEQ IDs are those which encode GBS1 to GBS689 (see Table IV of reference 3).

Within (b), the degree of sequence identity may vary depending on the amino acid sequence (a) in question, but is preferably greater than 50% (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more). Polypeptides within (b) include homologs, orthologs, allelic variants and functional mutants of (a). Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.

Within (c), the length of the fragment may vary depending on the amino acid sequence (a) in question, but the fragment is preferably at least 7 consecutive amino acids from the sequences of (a) e.g. 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more. Preferably the fragment comprises one or more epitopes from the sequence. Other preferred fragments are the N-terminal signal peptides of SEQ IDs 1-10966 from Ref. 3, SEQ IDs 1-10966 from Ref. 3 without their N-terminal signal peptides, and SEQ IDs 1-10966 from Ref. 3 wherein up to 10 amino acid residues (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues) are deleted from the N-terminus and/or the C-terminus e.g. the N-terminal amino acid residue may be deleted.

The polypeptides can, of course, be prepared by various means (e.g. recombinant expression, purification from GBS, chemical synthesis etc.) and in various forms (e.g. native, fusions, glycosylated, non-glycosylated etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other streptococcal or host cell proteins) or substantially isolated form.

Preferred polypeptide antigens are: GBS 80, GBS 91, GBS 104, GBS 147, GBS 173, GBS 276, GBS 305, GBS 313, GBS 322, GBS 328, GBS 330, GBS 338, GBS 358, GBS 361, GBS 404, GBS 656, GBS 690, and GBS 691, including polypeptides having amino acid sequences with sequence identity thereto etc.

The nucleotide and amino acid sequences of GBS80 in Ref. 3 are SEQ ID 8779 and SEQ ID 8780. These sequences are set forth below as SEQ ID NOS 1 and 2:

SEQ ID NO. 1 ATGAAATTATCGAAGAAGTTATTGTTTTCGGCTGCTGTTTTAACAATGGTGGCGGGGTCAACTGTTGAACCAGTAGCTCAGTTTGC GACTGGAATGAGTATTGTAAGAGCTGCAGAAGTGTCACAAGAACGCCCAGCGAAAACAACAGTAAATATCTATAAATTACAAGCTG ATAGTTATAAATCGGAAATTACTTCTAATGGTGGTATCGAGAATAAAGACGGCGAAGTAATATCTAACTATGCTAAACTTGGTGAC AATGTAAAAGGTTTGCAAGGTGTACAGTTTAAACGTTATAAAGTCAAGACGGATATTTCTGTTGATGAATTGAAAAAATTGACAAC AGTTGAAGCAGCAGATGCAAAAGTTGGAACGATTCTTGAAGAAGGTGTCAGTCTACCTCAAAAAACTAATGCTCAAGGTTTGGTCG TCGATGCTCTGGATTCAAAAAGTAATGTGAGATACTTGTATGTAGAAGATTTAAAGAATTCACCTTCAAACATTACCAAAGCTTAT GCTGTACCGTTTGTGTTGGAATTACCAGTTGCTAACTCTACAGGTACAGGTTTCCTTTCTGAAATTAATATTTACCCTAAAAACGT TGTAACTGATGAACCAAAAACAGATAAAGATGTTAAAAAATTAGGTCAGGACGATGCAGGTTATACGATTGGTGAAGAATTCAAAT GGTTCTTGAAATCTACAATCCCTGCCAATTTAGGTGACTATGAAAAATTTGAAATTACTGATAAATTTGCAGATGGCTTGACTTAT AAATCTGTTGGAAAAATCAAGATTGGTTCGAAAACACTGAATAGAGATGAGCACTACACTATTGATGAACCAACAGTTGATAACCA AAATACATTAAAAATTACGTTTAAACCAGAGAAATTTAAAGAAATTGCTGAGCTACTTAAAGGAATGACCCTTGTTAAAAATCAAG ATGCTCTTGATAAAGCTACTGCAAATACAGATGATGCGGCATTTTTGGAAATTCCAGTTGCATCAACTATTAATGAAAAAGCAGTT TTAGGAAAAGCAATTGAAAATACTTTTGAACTTCAATATGACCATACTCCTGATAAAGCTGACAATCCAAAACCATCTAATCCTCC AAGAAAACCAGAAGTTCATACTGGTGGGAAACGATTTGTAAAGAAAGACTCAACAGAAACACAAACACTAGGTGGTGCTGAGTTTG ATTTGTTGGCTTCTGATGGGACAGCAGTAAAATGGACAGATGCTCTTATTAAAGCGAATACTAATAAAAACTATATTGCTGGAGAA GCTGTTACTGGGCAACCAATCAAATTGAAATCACATACAGACGGTACGTTTGAGATTAAAGGTTTGGCTTATGCAGTTGATGCGAA TGCAGAGGGTACAGCAGTAACTTACAAATTAAAAGAAACAAAAGCACCAGAAGGTTATGTAATCCCTGATAAAGAAATCGAGTTTA CAGTATCACAAACATCTTATAATACAAAACCAACTGACATCACGGTTGATAGTGCTGATGCAACACCTGATACAATTAAAAACAAC AAACGTCCTTCAATCCCTAATACTGGTGGTATTGGTACGGCTATCTTTGTCGCTATCGGTGCTGCGGTGATGGCTTTTGCTGTTAA GGGGATGAAGCGTCGTACAAAAGATAAC SEQ ID NO: 2 MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGD NVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAY AVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTY KSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAV LGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGE AVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNN KRPSIPNTGGIGTAIFVAIGAAVMAFAVKGMKRRTKDN

The nucleotide and amino acid sequences of GBS 91 in Ref. 3 are SEQ ID 8937 and SEQ ID 8938. These sequences are set forth below as SEQ ID NOS 3 and 4:

SEQ ID NO. 3 ATGAAAAAAGGACAAGTAAATGATACTAAGCAATCTTACTCTCTACGTAAATATAAATTTGGTTTAGCATCAGTAATTTTAGGGTC ATTCATAATGGTCACAAGTCCTGTTTTTGCGGATCAAACTACATCGGTTCAAGTTAATAATCAGACAGGCACTAGTGTGGATGCTA ATAATTCTTCCAATGAGACAAGTGCGTCAAGTGTGATTACTTCCAATAATGATAGTGTTCAAGCGTCTGATAAAGTTGTAAATAGT CAAAATACGGCAACAAAGGACATTACTACTCCTTTAGTAGAGACAAAGCCAATGGTGGAAAAAACATTACCTGAACAAGGGAATTA TGTTTATAGCAAAGAAACCGAGGTGAAAAATACACCTTCAAAATCAGCCCCAGTAGCTTTCTATGCAAAGAAAGGTGATAAAGTTT TCTATGACCAAGTATTTAATAAAGATAATGTGAAATGGATTTCATATAAGTCTTTTTGTGGCGTACGTCGATACGCAGCTATTGAG TCACTAGATCCATCAGGAGGTTCAGAGACTAAAGCACCTACTCCTGTAACAAATTCAGGAAGCAATAATCAAGAGAAAATAGCAAC GCAAGGAAATTATACATTTTCACATAAAGTAGAAGTAAAAAATGAAGCTAAGGTAGCGAGTCCAACTCAATTTACATTGGACAAAG GAGACAGAATTTTTTACGACCAAATACTAACTATTGAAGGAAATCAGTGGTTATCTTATAAATCATTCAATGGTGTTCGTCGTTTT GTTTTGCTAGGTAAAGCATCTTCAGTAGAAAAAACTGAAGATAAAGAAAAAGTGTCTCCTCAACCACAAGCCCGTATTACTAAAAC TGGTAGACTGACTATTTCTAACGAAACAACTACAGGTTTTGATATTTTAATTACGAATATTAAAGATGATAACGGTATCGCTGCTG TTAAGGTACCGGTTTGGACTGAACAAGGAGGGCAAGATGATATTAAATGGTATACAGCTGTAACTACTGGGGATGGCAACTACAAA GTAGCTGTATCATTTGCTGACCATAAGAATGAGAAGGGTCTTTATAATATTCATTTATACTACCAAGAAGCTAGTGGGACACTTGT AGGTGTAACAGGAACTAAAGTGACAGTAGCTGGAACTAATTCTTCTCAAGAACCTATTGAAAATGGTTTAGCAAAGACTGGTGTTT ATAATATTATCGGAAGTACTGAAGTAAAAAATGAAGCTAAAATATCAAGTCAGACCCAATTTACTTTAGAAAAAGGTGACAAAATA AATTATGATCAAGTATTGACAGCAGATGGTTACCAGTGGATTTCTTACAAATCTTATAGTGGTGTTCGTCGCTATATTCCTGTGAA AAAGCTAACTACAAGTAGTGAAAAAGCGAAAGATGAGGCGACTAAACCGACTAGTTATCCCAACTTACCTAAAACAGGTACCTATA CATTTACTAAAACTGTAGATGTGAAAAGTCAACCTAAAGTATCAAGTCCAGTGGAATTTAATTTTCAAAAGGGTGAAAAAATACAT TATGATCAAGTGTTAGTAGTAGATGGTCATCAGTGGATTTCATACAAGAGTTATTCCGGTATTCGTCGCTATATTGAAATT SEQ ID NO. 4 MKKGQVNDTKQSYSLRKYKFGLASVILGSFIMVTSPVFADQTTSVQVNNQTGTSVDANNSSNETSASSVITSNNDSVQASDKVVNS QNTATKDITTPLVETKPMVEKTLPEQGNYVYSKETEVKNTPSKSAPVAFYAKKGDKVFYDQVFNKDNVKWISYKSFCGVRRYAAIE SLDPSGGSETKAPTPVTNSGSNNQEKIATQGNYTFSHKVEVKNEAKVASPTQFTLDKGDRIFYDQILTIEGNQWLSYKSFNGVRRF VLLGKASSVEKTEDKEKVSPQPQARITKTGRLTISNETTTGFDILITNIKDDNGIAAVKVPVWTEQGGQDDIKWYTAVTTGDGNYK VAVSFADHKNEKGLYNIHLYYQEASGTLVGVTGTKVTVAGTNSSQEPIENGLAKTGVYNIIGSTEVKNEAKISSQTQFTLEKGDKI NYDQVLTADGYQWISYKSYSGVRRYIPVKKLTTSSEKAKDEATKPTSYPNLPKTGTYTFTKTVDVKSQPKVSSPVEFNFQKGEKIH YDQVINVDGHQWISYKSYSGIRRYIEI

The nucleotide and amino acid sequences of GBS 104 in Ref. 3 are SEQ ID 8777 and SEQ ID 8778. These sequences are set forth below as SEQ ID NOS 5 and 6:

SEQ ID NO. 5 ATGAAAAAGAGACAAAAAATATGGAGAGGGTTATCAGTTACTTTACTAATCCTGTCCCAAATTCCATTTGGTATATTGGTACAAGG TGAAACCCAAGATACCAATCAAGCACTTGGAAAAGTAATTGTTAAAAAAACGGGAGACAATGCTACACCATTAGGCAAAGCGACTT TTGTGTTAAAAAATGACAATGATAAGTCAGAAACAAGTCACGAAACGGTAGAGGGTTCTGGAGAAGCAACCTTTGAAAACATAAAA CCTGGAGACTACACATTAAGAGAAGAAACAGCACCAATTGGTTATAAAAAAACTGATAAAACCTGGAAAGTTAAAGTTGCAGATAA CGGAGCAACAATAATCGAGGGTATGGATGCAGATAAAGCAGAGAAACGAAAAGAAGTTTTGAATGCCCAATATCCAAAATCAGCTA TTTATGAGGATACAAAAGAAAATTACCCATTAGTTAATGTAGAGGGTTCCAAAGTTGGTGAACAATACAAAGCATTGAATCCAATA AATGGAAAAGATGGTCGAAGAGAGATTGCTGAAGGTTGGTTATCAAAAAAAATTACAGGGGTCAATGATCTCGATAAGAATAAATA TAAAATTGAATTAACTGTTGAGGGTAAAACCACTGTTGAAACGAAAGAACTTAATCAACCACTAGATGTCGTTGTGCTATTAGATA ATTCAAATAGTATGAATAATGAAAGAGCCAATAATTCTCAAAGAGCATTAAAAGCTGGGGAAGCAGTTGAAAAGCTGATTGATAAA ATTACATCAAATAAAGACAATAGAGTAGCTCTTGTGACATATGCCTCAACCATTTTTGATGGTACTGAAGCGACCGTATCAAAGGG AGTTGCCGATCAAAATGGTAAAGCGCTGAATGATAGTGTATCATGGGATTATCATAAAACTACTTTTACAGCAACTACACATAATT ACAGTTATTTAAATTTAACAAATGATGCTAACGAAGTTAATATTCTAAAGTCAAGAATTCCAAAGGAAGCGGAGCATATAAATGGG GATCGCACGCTCTATCAATTTGGTGCGACATTTACTCAAAAAGCTCTAATGAAAGCAAATGAAATTTTAGAGACACAAAGTTCTAA TGCTAGAAAAAAACTTATTTTTCACGTAACTGATGGTGTCCCTACGATGTCTTATGCCATAAATTTTAATCCTTATATATCAACAT CTTACCAAAACCAGTTTAATTCTTTTTTAAATAAAATACCAGATAGAAGTGGTATTCTCCAAGAGGATTTTATAATCAATGGTGAT GATTATCAAATAGTAAAAGGAGATGGAGAGAGTTTTAAACTGTTTTCGGATAGAAAAGTTCCTGTTACTGGAGGAACGACACAAGC AGCTTATCGAGTACCGCAAAATCAACTCTCTGTAATGAGTAATGAGGGATATGCAATTAATAGTGGATATATTTATCTCTATTGGA GAGATTACAACTGGGTCTATCCATTTGATCCTAAGACAAAGAAAGTTTCTGCAACGAAACAAATCAAAACTCATGGTGAGCCAACA ACATTATACTTTAATGGAAATATAAGACCTAAAGGTTATGACATTTTTACTGTTGGGATTGGTGTAAACGGAGATCCTGGTGCAAC TCCTCTTGAAGCTGAGAAATTTATGCAATCAATATCAAGTAAAACAGAAAATTATACTAATGTTGATGATACAAATAAAATTTATG ATGAGCTAAATAAATACTTTAAAACAATTGTTGAGGAAAAACATTCTATTGTTGATGGAAATGTGACTGATCCTATGGGAGAGATG ATTGAATTCCAATTAAAAAATGGTCAAAGTTTTACACATGATGATTACGTTTTGGTTGGAAATGATGGCAGTCAATTAAAAAATGG TGTGGCTCTTGGTGGACCAAACAGTGATGGGGGAATTTTAAAAGATGTTACAGTGACTTATGATAAGACATCTCAAACCATCAAAA TCAATCATTTGAACTTAGGAAGTGGACAAAAAGTAGTTCTTACCTATGATGTACGTTTAAAAGATAACTATATAAGTAACAAATTT TACAATACAAATAATCGTACAACGCTAAGTCCGAAGAGTGAAAAAGAACCAAATACTATTCGTGATTTCCCAATTCCCAAAATTCG TGATGTTCGTGAGTTTCCGGTACTAACCATCAGTAATCAGAAGAAAATGGGTGAGGTTGAATTTATTAAAGTTAATAAAGACAAAC ATTCAGAATCGCTTTTGGGAGCTAAGTTTCAACTTCAGATAGAAAAAGATTTTTCTGGGTATAAGCAATTTGTTCCAGAGGGAAGT GATGTTACAACAAAGAATGATGGTAAAATTTATTTTAAAGCACTTCAAGATGGTAACTATAAATTATATGAAATTTCAAGTCCAGA TGGCTATATAGAGGTTAAAACGAAACCTGTTGTGACATTTACAATTCAAAATGGAGAAGTTACGAACCTGAAAGCAGATCCAAATG CTAATAAAAATCAAATCGGGTATCTTGAAGGAAATGGTAAACATCTTATTACCAACACTCCCAAACGCCCACCAGGTGTTTTTCCT AAAACAGGGGGAATTGGTACAATTGTCTATATATTAGTTGGTTCTACTTTTATGATACTTACCATTTGTTCTTTCCGTCGTAAACA ATTG SEQ ID NO. 6 MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEATFENIK PGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPI NGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGEAVEKLIDK ITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHING DRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGD DYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPT TLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEM IEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTYDVRLKDNYISNKF YNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGS DVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANENQIGYLEGNGKHLITNTPKRPPGVFP KTGGIGTIVYILVGSTFMILTICSFRRKQL

The nucleotide and amino acid sequences of GBS 147 in Ref. 3 are SEQ ID 8525 and SEQ ID 8526. These sequences are set forth below as SEQ ID NOS 7 and 8:

SEQ ID NO. 7 GTGGATAAACATCACTCAAAAAAGGCTATTTTAAAGTTAACACTTATAACAACTAGTATTTTATTAATGCATAGCAATCAAGTGAATGCAGAGGAG CAAGAATTAAAAAACCAAGAGCAATCACCTGTAATTGCTAATGTTGCTCAACAGCCATCGCCATCGGTAACTACTAATACTGTTGAAAAAACATCT GTAACAGCTGCTTCTGCTAGTAATACAGCGAAAGAAATGGGTGATACATCTGTAAAAAATGACAAAACAGAAGATGAATTATTAGAAGAGTTATCT AAAAACCTTGATACGTCTAATTTGGGGGCTGATCTTGAAGAAGAATATCCCTCTAAACCAGAGACAACCAACAATAAAGAAAGCAATGTAGTAACA AATGCTTCAACTGCAATAGCACAGAAAGTTCCCTCAGCATATGAAGAGGTGAAGCCAGAAAGCAAGTCATCGCTTGCTGTTCTTGATACATCTAAA ATAACAAAATTACAAGCCATAACCCAAAGAGGAAAGGGAAATGTAGTAGCTATTATTGATACTGGCTTTGATATTAACCATGATATTTTTCGTTTA GATAGCCCAAAAGATGATAAGCACAGCTTTAAAACTAAGACAGAATTTGAGGAATTAAAAGCAAAACATAATATCACTTATGGGAAATGGGTTAAC GATAAGATTGTTTTTGCACATAACTACGCCAACAATACAGAAACGGTGGCTGATATTGCAGCAGCTATGAAAGATGGTTATGGTTCAGAAGCAAAG AATATTTCGCATGGTACACACGTTGCTGGTATTTTTGTAGGTAATAGTAAACGTCCAGCAATCAATGGTCTTCTTTTAGAAGGTGCAGCGCCAAAT GCTCAAGTCTTATTAATGCGTATTCCAGATAAAATTGATTCGGACAAATTTGGTGAAGCATATGCTAAAGCAATCACAGACGCTGTTAATCTAGGA GCAAAAACGATTAATATGAGTATTGGAAAAACAGCTGATTCTTTAATTGCTCTCAATGATAAAGTTAAATTAGCACTTAAATTAGCTTCTGAGAAG GGCGTTGCAGTTGTTGTGGCTGCCGGAAATGAAGGCGCATTTGGTATGGATTATAGCAAACCATTATCAACTAATCCTGACTACGGTACGGTTAAT AGTCCAGCTATTTCTGAAGATACTTTGAGTGTTGCTAGCTATGAATCACTTAAAACTATCAGTGAGGTCGTTGAAACAACTATTGAAGGTAAGTTA GTTAAGTTGCCGATTGTGACTTCTAAACCTTTTGACAAAGGTAAGGCCTACGATGTGGTTTATGCCAATTATGGTGCAAAAAAAGACTTTGAAGGT AAGGACTTTAAAGGTAAGATTGCATTAATTGAGCGTGGTGGTGGACTTGATTTTATGACTAAAATCACTCATGCTACAAATGCAGGTGTTGTTGGT ATCGTTATTTTTAACGATCAAGAAAAACGTGGAAATTTTCTAATTCCTTACCGTGAATTACCTGTGGGGATTATTAGTAAAGTAGATGGCGAGCGT ATAAAAAATACTTCAAGTCAGTTAACATTTAACCAGAGTTTTGAAGTAGTTGATAGCCAAGGTGGTAATCGTATGCTGGAACAATCAAGTTGGGGC GTGACAGCTGAAGGAGCAATCAAGCCTGATGTAACAGCTTCTGGCTTTGAAATTTATTCTTCAACCTATAATAATCAATACCAAACAATGTCTGGT ACAAGTATGGCTTCACCACATGTTGCAGGATTAATGACAATGCTTCAAAGTCATTTGGCTGAGAAATATAAAGGGATGAATTTAGATTCTAAAAAA TTGCTAGAATTGTCTAAAAACATCCTCATGAGCTCAGCAACAGCATTATATAGTGAAGAGGATAAGGCGTTTTATTCACCACGTCAGCAAGGTGCA GGTGTAGTTGATGCTGAAAAAGCTATCCAAGCTCAATATTATATTACTGGAAACGATGGCAAAGCTAAAATTAATCTCAAACGAATGGGAGATAAA TTTGATATCACAGTTACAATTCATAAACTTGTAGAAGGTGTCAAAGAATTGTATTATCAAGCTAATGTAGCAACAGAACAAGTAAATAAAGGTAAA TTTGCCCTTAAACCACAAGCCTTGCTAGATACTAATTGGCAGAAAGTAATTCTTCGTGATAAAGAAACACAAGTTCGATTTACTATTGATGCTAGT CAATTTAGTCAGAAATTAAAAGAACAGATGGCAAATGGTTATTTCTTAGAAGGTTTTGTACGTTTTAAAGAAGCCAAGGATAGTAATCAGGAGTTA ATGAGTATTCCTTTTGTAGGATTTAATGGTGATTTTGCGAACTTACAAGCACTTGAAACACCGATTTATAAGACGCTTTCTAAAGGTAGTTTCTAC TATAAACCAAATGATACAACTCATAAAGACCAATTGGAGTACAATGAATCAGCTCCTTTTGAAAGCAACAACTATACTGCCTTGTTAACACAATCA GCGTCTTGGGGCTATGTTGATTATGTCAAAAATGGTGGGGAGTTAGAATTAGCACCGGAGAGTCCAAAAAGAATTATTTTAGGAACTTTTGAGAAT AAGGTTGAGGATAAAACAATTCATCTTTTGGAAAGAGATGCAGCGAATAATCCATATTTTGCCATTTCTCCAAATAAAGATGGAAATAGGGACGAA ATCACTCCCCAGGCAACTTTCTTAAGAAATGTTAAGGATATTTCTGCTCAAGTTCTAGATCAAAATGGAAATGTTATTTGGCAAAGTAAGGTTTTA CCATCTTATCGTAAAAATTTCCATAATAATCCAAAGCAAAGTGATGGTCATTATCGTATGGATGCTCTTCAGTGGAGTGGTTTAGATAAGGATGGC AAAGTTGTAGCAGATGGTTTTTATACTTATCGCTTACGTTACACACCAGTAGCAGAAGGAGCAAATAGTCAGGAGTCAGACTTTAAAGTACAAGTA AGTACTAAGTCACCAAATCTTCCTTCACGAGCTCAGTTTGATGAAACTAATCGAACATTAAGCTTAGCCATGCCTAAGGAAAGTAGTTATGTTCCT ACATATCGTTTACAATTAGTTTTATCTCATGTTGTAAAAGATGAAGAATATGGGGATGAGACTTCTTACCATTATTTCCATATAGATCAAGAAGGT AAAGTGACACTTCCTAAAACGGTTAAGATAGGAGAGAGTGAGGTTGCGGTAGACCCTAAGGCCTTGACACTTGTTGTGGAAGATAAAGCTGGTAAT TTCGCAACGGTAAAATTGTCTGATCTCTTGAATAAGGCAGTAGTATCAGAGAAAGAAAACGCTATAGTAATTTCTAACAGTTTCAAATATTTTGAT AACTTGAAAAAAGAACCTATGTTTATTTCTAAAAAAGAAAAAGTAGTAAACAAGAATCTAGAAGAAATAATATTAGTTAAGCCGCAAACTACAGTT ACTACTCAATCATTGTCTAAAGAAATAACTAAATCAGGAAATGAGAAAGTCCTCACTTCTACAAACAATAATAGTAGCAGAGTAGCTAAGATCATA TCACCTAAACATAACGGGGATTCTGTTAACCATACCTTACCTAGTACATCAGATAGAGCAACGAATGGTCTATTTGTTGGTACTTTGGCATTGTTA TCTAGTTTACTTCTTTATTTGAAACCCAAAAAGACTAAAAATAATAGTAAA SEQ ID NO. 8 VDKHHSKKAILKLTLITTSILLMHSNQVNAEEQELKNQEQSPVIANVAQQPSPSVTTNTVEKTSVTAASASNTAKEMGDTSVKNDKTEDELLEELS ENLDTSNLGADLEEEYPSKPETTNNKESNVVTNASTAIAQKVPSAYEEVKPESKSSLAVLDTSKITKLQAITQRGKGNVVAIIDTGFDINHDIFRL DSPKDDKHSFKTKTEFEELKAKHNITYGKNVNDKIVFAHNYANNTETVADIAAAMKDGYGSEAKNISHGTHVAGIFVGNSKRPAINGLLLEGAAPN AQVLLMRIPDKIDSDKFGEAYAKAITDAVNLGAKTINMSIGKTADSLIALNDKVKLALKLASEKGVAVVVAAGNEGAFGMDYSKPLSTNPDYGTVN SPAISEDTLSVASYESLKTISEVVETTIEGKLVKLPIVTSKPFDKGKAYDVVYANYGAKKDPEGKDFKGKIALIERGGGLDFMTKITHATNAGVVG IVIFNDQEKRGNFLIPYRELPVGIISKVDGERIKNTSSQLTFNQSFEVVDSQGGNRMLEQSSWGVTAEGAIKPDVTASGFEIYSSTYNNQYQTMSG TSMASPHVAGLMTMLQSHLAEKYKGMNLDSKKLLELSKNILMSSATALYSEEDKAFYSPRQQGAGVVDAEKAIQAQYYITGNDGKAKINLKRMGDK FDITVTIHKLVEGVKELYYQANVATEQVNKGKFALKPQALLDTNWQKVILRDKETQVRFTIDASQFSQKLKEQMANGYFLEGFVRFKEAKDSNQHL MSIPFVGFNGDFANLQALETPIYKTLSKGSFYYKPNDTTHKDQLEYNESAPFESNNYTALLTQSASWGYVDYVKNGGELELAPESPKRIILGTFEN KVEDKTIHLLERDAANNPYFAISPNKDGNRDEITPQATFLRNVKDISAQVLDQNGNVIWQSKVLPSYRKNFHNNPKQSDGHYRMDALQWSGLDKDG KVVADGFYTYRLRYTPVAEGANSQESDFKVQVSTKSPNLPSRAQFDETNRTLSLAMPKESSYVPTYRLQLVLSHVVKDEEYGDETSYHYFHIDQEG KVTLPKTVKIGESEVAVDPKALTLVVEDKAGNFATVKLSDLLNKAVVSEKENAIVISNSFKYFDNLKKEPMFISKKEKVVNKNLEEIILVKPQTTV TTQSLSKEITKSGNEKVLTSTNNNSSRVAKIISPKHNGDSVNHTLPSTSDRATNGLFVGTLALLSSLLLYLKPKKTKNNSK

The nucleotide and amino acid sequences of GBS 173 in Ref. 3 are SEQ ID 8787 and SEQ ID 8788. These sequences are set forth below as SEQ ID NOS 9 and 10:

SEQ ID NO. 9 ATGAAACGTAAATACTTTATTCTTAATACGGTGACGGTTTTAACGTTAGCTGCTGCAATGAATACTAGCAGTATCTATGCTAATAGTACTGAGACA AGTGCTTCAGTAGTTCCTACTACAAATACTATCGTTCAAACTAATGACAGTAATCCTACCGCAAAATTTGTATCAGAATCAGGACAATCTGTAATA GGTCAAGTAAAACCAGATAATTCTGCGGCGCTTACAACAGTTGACACGCCTCATCATATTTCAGCTCCAGATGCTTTAAAAACAACTCAATCAAGT CCTGTCGTTGAGAGTACTTCTACTAAGTTAACTGAAGAGACTTACAAACAAAAAGATGGTCAAGATTTAGCCAACATGGTGAGAAGTGGTCAAGTT ACTAGTGAGGAACTCGTTAATATGGCATACGATATTATTGCTAAAGAAAACCCATCTTTAAATGCAGTCATTACTACTAGACGCCAAGAAGCTATT GAAGAGGCTAGAAAACTTAAAGATACCAATCAGCCGTTTTTAGGTGTTCCCTTGTTAGTCAAGGGGTTAGGGCACAGTATTAAAGGTGGTGAAACC AATAATGGCTTGATCTATGCAGATGGAAAAATTAGCACATTTGACAGTAGCTATGTCAAAAAATATAAAGATTTAGGATTTATTATTTTAGGACAA ACGAACTTTCCAGAGTATGGGTGGCGTAATATAACAGATTCTAAATTATACGGTCTAACGCATAATCCTTGGGATCTTGCTCATAATGCTGGTGGC TCTTCTGGTGGAAGTGCAGCAGCCATTGCTAGCGGAATGACGCCAATTGCTAGCGGTAGTGATGCTGGTGGTTCTATCCGTATTCCATCTTCTTGG ACGGGCTTGGTAGGTTTAAAACCAACAAGAGGATTGGTGAGTAATGAAAAGCCAGATTCGTATAGTACAGCAGTTCATTTTCCATTAACTAAGTCA TCTAGAGACGCAGAAACATTATTAACTTATCTAAAGAAAAGCGATCAAACGCTAGTATCAGTTAATGATTTAAAATCTTTACCAATTGCTTATACT TTGAAATCACCAATGGGAACAGAAGTTAGTCAAGATGCTAAAAACGCTATTATGGACAACGTCACATTCTTAAGAAAACAAGGATTCAAAGTAACA GAGATAGACTTACCAATTGATGGTAGAGCATTAATGCGTGATTATTCAACCTTGGCTATTGGCATGGGAGGAGCTTTTTCAACAATTGAAAAAGAC TTAAAAAAACATGGTTTTACTAAAGAAGACGTTGATCCTATTACTTGGGCAGTTCATGTTATTTATCAAAATTCAGATAAGGCTGAACTTAAGAAA TCTATTATGGAAGCCCAAAAACATATGGATGATTATCGTAAGGCAATGGAGAAGCTTCACAAGCAATTTCCTATTTTCTTATCGCCAACGACCGCA AGTTTAGCCCCTCTAAATACAGATCCATATGTAACAGAGGAAGATAAAAGAGCGATTTATAATATGGAAAACTTGAGCCAAGAAGAAAGAATTGCT CTCTTTAATCGCCAGTGGGAGCCTATGTTGCGTAGAACACCTTTTACACAAATTGCTAATATGACAGGACTCCCAGCTATCAGTATCCCGACTTAC TTATCTGAGTCTGGTTTACCCATAGGGACGATGTTAATGGCAGGTGCAAACTATGATATGGTATTAATTAAATTTGCAACTTTCTTTGAAAAACAT CATGGTTTTAATGTTAAATGGCAAAGAATAATAGATAAAGAAGTGAAACCATCTACTGGCCTAATACAGCCTACTAACTCCCTCTTTAAAGCTCAT TCATCATTAGTAAATTTAGAAGAAAATTCACAAGTTACTCAAGTATCTATCTCTAAAAAATGGATGAAATCGTCTGTTAAAAATAAACCATCCGTA ATGGCATATCAAAAAGCACTTCCTAAAACAGGTGATACAGAATCAAGCCTATCTCCAGTTTTAGTAGTAACCCTTTTATTAGCTTGTTTTAGCTTT GTAACAAAAAAGAATCAGAAAAGT SEQ ID NO. 10 MKRKYFILNTVTVLTLAAAMNTSSIYANSTETSASVVPTTNTIVQTNDSNPTAKFVSESGQSVIGQVKPDNSAALTTVDTPHHISAPDALKTTQSS PVVESTSTKLTEETYKQKDGQDLANMVRSGQVTSEELVNMAYDIIAKENPSLNAVITTRRQEAIEEARKLKDTNQPFLGVPLLVKGLGHSIKGGET NNGLIYADGKISTFDSSYVKKYKDLGFIILGQTNFPEYGWRNITDSKLYGLTHNPWDLAHNAGGSSGGSAAAIASGMTPIASGSDAGGSIRIPSSW TGLVGLKPTRGLVSNEKPDSYSTAVHFPLTKSSRDAETLLTYLKKSDQTLVSVNDLKSLPIAYTLKSPMGTEVSQDAKNAIMDNVTFLRKQGFKVT EIDLPIDGRALMRDYSTLAIGMGGAFSTIEKDLKKHGFTKEDVDPITWAVHVIYQNSDKAELKKSIMEAQKHMDDYRKAMEKLHKQFPIFLSPTTA SLAPLNTDPYVTEEDKRAIYNMENLSQEERIALFNRQWEPMLRRTPFTQIANMTGLPAISIPTYLSESGLPIGTMLMAGANYDMVLIKFATFFEKH HGFNVKWQRIIDKEVKPSTGLIQPTNSLFKAHSSLVNLEENSQVTQVSISKKWMKSSVKNKPSVMAYQKALPKTGDTESSLSPVLVVTLLLACFSF VTKKNQKS

The nucleotide and amino acid sequences of GBS 276 in Ref. 3 are SEQ ID 8941 and SEQ ID 8942. These sequences are set forth below as SEQ ID NOS 11 and 12:

SEQ ID NO. 11 TTGCGTAAAAAACAAAAACTACCATTTGATAAACTTGCCATTGCGCTTATATCTACGAGCATCTTGCTCAATGCACAATCAGACATTAAAGCAAAT ACTGTGACAGAAGACACTCCTGCTACCGAACAAGCCGTAGAACCCCCACAACCAATAGCAGTTTCTGAGGAATCACGATCATCAAAGGAAACTAAA ACCTCACAAACTCCTAGTGATGTAGGAGAAACAGTAGCAGATGACGCTAATGATCTAGCCCCTCAAGCTCCTGCTAAAACTGCTGATACACCAGCA ACCTCAAAAGCGACTATTAGGGATTTGAACGACCCTTCTCATGTCAAAACCCTGCAGGAAAAAGCAGGCAAGGGAGCTGGGACCGTTGTTGCAGTG ATTGATGCTGGTTTTGATAAAAATCATGAAGCGTGGCGCTTAACAGACAAAACTAAAGCACGTTACCAATCAAAAGAAAATCTTGAAAAAGCTAAA AAAGAGCACGGTATTACCTATGGCGAGTGGGTCAATGATAAGGTTGCTTATTACCACGACTATAGTAAAGATGGTAAAAACGCTGTTGATCAAGAA CACGGCACACACGTGTCAGGGATCTTGTCAGGAAATGCTCCATCTGAAATGAAAGAACCTTACCGCCTAGAAGGTGCGATGCCTGAGGCTCAATTG CTTTTGATGCGTGTCGAAATTGTAAATGGACTAGCAGACTATGCTCGTAACTACGCTCAAGCTATCAGAGATGCTGTCAACTTGGGAGCTAAGGTG ATTAATATGAGCTTTGGTAATGCTGCACTAGCTTACGCCAACCTTCCAGACGAAACCAAAAAAGCCTTTGACTATGCCAAATCAAAAGGTGTTAGC ATTGTGACCTCAGCTGGTAATGATAGTAGCTTTGGGGGCAAGCCCCGTCTACCTCTAGCAGATCATCCTGATTATGGGGTGGTTGGGACACCTGCA GCGGCAGATTCAACATTGACAGTTGCTTCTTACAGCCCAGATAAACAGCTCACTGAAACTGCTACGGTCAAAACAGACGATCATCAAGATAAAGAA ATGCCTGTTATTTCAACAAACCGTTTTGAGCCAAACAAGGCTTACGACTATGCTTATGCTAATCGTGGTACGAAAGAGGATGATTTTAAGGATGTC GAAGGTAAGATTGCCCTTATTGAACGTGGCGATATTGATITCAAAGATAAGATTGCAAACGCTAAAAAAGCTGGTGCTGTAGGGGTCTTGATCTAT GACAATCAAGACAAGGGCTTCCCGATTGAATTGCCAAATGTTGACCAGATGCCTGCGGCCTTTATCAGTCGAAGAGACGGTCTCTTATTAAAAGAC AATCCCCCAAAAACCATTACCTTCAATGCGACACCTAAGGTATTGCCAACAGCAAGTGGCACCAAACTAAGCCGCTTCTCAAGCTGGGGTCTGACA GCTGACGGCAATATTAAACCGGATATTGCAGCACCCGGCCAAGATATTTTGTCATCAGTGGCTAACAACAAGTATGCCAAACTTTCTGGAACTAGT ATGTCTGCACCATTGGTAGCGGGTATCATGGGACTGTTGCAAAAGCAATATGAGACACAGTATCCTGATATGACACCATCAGAGCGTCTTGATTTA GCTAAGAAAGTATTGATGAGCTCAGCAACTGCCCTATATGATGAAGATGAAAAAGCTTATTTTTCTCCTCGCCAACAGGGAGCAGGAGCAGTCGAT GCTAAAAAAGCTTCAGCAGCAACGATGTATGTAACAGATAAGGACAATACCTCAAGCAAGGTTCACCTGAACAATGTTTCTGATAAATTTGAAGTA ACAGTAACAGTTCACAACAAATCTGATAAACCTCAAGAGTTGTATTACCAAGTAACTGTTCAAACAGATAAAGTAGATGGAAAACACTTTGCCTTG GCTCCTAAAGCATTGTATGAGACATCATGGCAAAAAATCACAATTCCAGCCAATAGCAGCAAACAAGTCACCGTTCCAATCGATGCTAGTCGATTT AGCAAGGACTTGCTTGCCCAAATGAAAAATGGCTATTTCTTAGAAGGTTTTGTTCGTTTCAAACAAGATCCTACAAAAGAAGAGCTTATGAGCATT CCATATATTGGTTTCCGAGGTGATTTTGGCAATCTGTCAGCCTTAGAAAAACCAATCTATGATAGCAAAGACGGTAGCAGCTACTATCATGAAGCA AATAGTGATGCCAAAGACCAATTAGATGGTGATGGATTACAGTTTTACGCTCTGAAAAATAACTTTACAGCACTTACCACAGAGTCTAACCCATGG ACGATTATTAAAGCTGTCAAAGAAGGGGTTGAAAACATAGAGGATATCGAATCTTCAGAGATCACAGAAACCATTTTTGCAGGTACTTTTGCAAAA CAAGACGATGATAGCCACTACTATATCCACCGTCACGCTAATGGCAAACCATATGCTGCGATCTCTCCAAATGGGGACGGTAACAGAGATTATGTC CAATTCCAAGGTACTTTCTTGCGTAATGCTAAAAACCTTGTGGCTGAAGTCTTGGACAAAGAAGGAAATGTTGTTTGGACAAGTGAGGTAACCGAG CAAGTTGTTAAAAACTACAACAATGACTTGGCAAGCACACTTGGTTCAACCCGTTTTGAAAAAACGCGTTGGGACGGTAAAGATAAAGACGGCAAA GTTGTTGCTAACGGAACCTACACCTATCGTGTTCGCTACACGCCGATTAGCTCAGGTGCAAAAGAACAACACACTGATTTTGATGTGATTGTAGAC AATACGACACCTGAAGTCGCAACATCGGCAACATTCTCAACAGAAGATAGTCGTTTGACACTTGCATCTAAACCAAAAACCAGCCAACCGGTTTAC CGTGAGCGTATTGCTTACACTTATATGGATGAGGATCTGCCAACAACAGAGTATATTTCTCCAAATGAAGATGGTACCTTTACTCTTCCTGAAGAG GCTGAAACAATGGAAGGCGCTACTGTTCCATTGAAAATGTCAGACTTTACTTATGTTGTTGAAGATATGGCTGGTAACATCACTTATACACCAGTG ACTAAGCTATTGGAGGGCCACTCTAATAAGCCAGAACAAGACGGTTCAGATCAAGCACCAGACAAGAAACCAGAAGCTAAACCAGAACAAGACGGT TCAGGTCAAACACCAGATAAAAAAAAAGAAACTAAACCAGAAAAAGATAGTTCAGGTCAAACACCAGGTAAAACTCCTCAAAAAGGTCAATCTTCT CGTACTCTAGAGAAACGATCTTCTAAGCGTGCTTTAGCTACAAAAGCATCAACAAGAGATCAGTTACCAACGACTAATGACAAGGATACAAATCGT TTACATCTCCTTAAGTTAGTTATGACCACTTTCTTCTTGGGA SEQ ID NO. 12 MRKKQKLPFEKLAIALISTSILLNAQSDIKANTVTEDTPATEQAVEPPQPIAVSEESRSSKETKTSQTPSDVGETVADDANDLAPQ APAKTADTPATSKATIRDLNDPSHVKTLQEKAGKGAGTVVAVIDAGFDKNHEAWRLTDKTKARYQSKENLEKAKKEHGTTYGEWVN DKVAYYHDYSKDGKNAVDQEHGTHVSGILSGNAPSEMKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKVIN MSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKPRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETATVK TDDHQDKEMPVISTNRFEPNKAYDYAYANRGTKEDDFKDVEGKIALIERGDIDFKDKIANAKKAGAVGVLIYDNQDKGFPIELPNV DQMPAAFISRRDGLLLKDNPPKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTSMSAP LVAGIMGLLQKQYETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLNNV SDKFEVTVTVHNKSDKPQELYYQVTVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEG FVRFKQDPTKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALENNFTALTTESNPWTIIKAV KEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTS EVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDSR LTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHS NKPEQDGSDQAPDKKPEAKPEQDGSGQTPDKKKETKPEKDSSGQTPGKTPQKGQSSRTLEKRSSKRALATKASTRDQLPTTNDKDT NRLHLLKLVMTTFFLG

The nucleotide and amino acid sequences of GBS 305 in Ref. 3 are SEQ ID 207 and SEQ ID 208. These sequences are set forth below as SEQ ID NOS 13 and 14:

SEQ ID NO. 13 ATGGGACGAGTAATGAAAACAATAACAACATTTGAAAATAAAAAAGTTTTAGTCCTTGGTTTAGCACGATCTGGAGAAGCTGCTGC ACGTTTGTTAGCTAAGTTAGGAGCAATAGTGACAGTTAATGATGGCAAACCATTTGATGAAAATCCAACAGCACAGTCTTTGTTGG AAGAGGGTATTAAAGTGGTTTGTGGTAGTCATCCTTTAGAATTGTTAGATGAGGATTTTTGTTACATGATTAAAAATCCAGGAATA CCTTATAACAATCCTATGGTCAAAAAAGCATTAGAAAAACAAATCCCTGTTTTGACTGAAGTGGAATTAGCATACTTAGTTTCAGA ATCTCAGCTAATAGGTATTACAGGCTCTAACGGGAAAACGACAACGACAACGATGATTGCAGAAGTCTTAAATGCTGGAGGTCAGA GAGGTTTGTTAGCTGGGAATATCGGCTTTCCTGCTAGTGAAGTTGTTCAGGCTGCGAATGATAAAGATACTCTAGTTATGGAATTA TCAAGTTTTCAGCTAATGGGAGTTAAGGAATTTCGTCCTCATATTGCAGTAATTACTAATTTAATGCCAACTCATTTAGATTATCA TGGGTCTTTTGAAGATTATGTTGCTGCAAAATGGAATATCCAAAATCAAATGTCTTCATCTGATTTTTTGGTACTTAATTTTAATC AAGGTATTTCTAAAGAGTTAGCTAAAACTACTAAAGCAACAATCGTTCCTTTCTCTACTACGGAAAAAGTTGATGGTGCTTACGTA CAAGACAAGCAACTTTTCTATAAAGGGGAGAATATTATGTCAGTAGATGACATTGGTGTCCCAGGAAGCCATAACGTAGAGAATGC TCTAGCAACTATTGCGGTTGCTAAACTGGCTGGTATCAGTAATCAAGTTATTAGAGAAACTTTAAGCAATTTTGGAGGTGTTAAAC ACCGCTTGCAATCACTCGGTAAGGTTCATGGTATTAGTTTCTATAACGACAGCAAGTCAACTAATATATTGGCAACTCAAAAAGCA TTATCTGGCTTTGATAATACTAAAGTTATCCTAATTGCAGGAGGTCTTGATCGCGGTAATGAGTTTGATGAATTGATACCAGATAT CACTGGACTTAAACATATGGTTGTTTTAGGGGAATCGGCATCTCGAGTAAAACGTGCTGCACAAAAAGCAGGAGTAACTTATAGCG ATGCTTTAGATGTTAGAGATGCGGTACATAAAGCTTATGAGGTGGCACAACAGGGCGATGTTATCTTGCTAAGTCCTGCAAATGCA TCATGGGACATGTATAAGAATTTCGAAGTCCGTGGTGATGAATTCATTGATACTTTCGAAAGTCTTAGAGGAGAG SEQ ID NO. 14 MGRVMKTITTFENKKVLVLGLARSGEAAARLLAKLGAIVTVNDGKPFDENPTAQSLLEEGIKVVCGSHPLELLDEDFCYMIKNPGI PYNNPMVKKALEKQIPVLTEVELAYLVSESQLIGITGSNGKTTTTTMIAEVLNAGGQRGLLAGNIGFPASEVVQAANDKDTLVMEL SSFQLMGVKEFRPHIAVITNLMPTHLDYHGSFEDYVAAKWNIQNQMSSSDFLVLNFNQGISKELAKTTKATIVPFSTTEKVDGAYV QDKQLFYKGENIMSVDDIGVPGSHNVENALATIAVAKLAGISNQVIRETLSNFGGVKHRLQSLGKVHGISFYNDSKSTNILATQKA LSGFDNTKVILIAGGLDRGNEFDELIPDITGLKHMVVLGESASRVKRAAQKAGVTYSDALDVRDAVHKAYEVAQQGDVILLSPANA SWDMYKNFEVRGDEFIDTFESLRGE

The nucleotide and amino acid sequences of GBS 313 are in Ref. 3 are SEQ ID 4089 and SEQ ID 4090. These sequences are set forth as SEQ ID NOS 15 and 16 below:

SEQ ID NO. 15 ATGAAACGTATTGCTGTTTTAACTAGTGGTGGTGACGCCCCTGGTATGAACGCTGCTATCCGTGCAGTTGTTCGTAAAGCAATTTCTGAAGGTATG GAAGTTTACGGCATCAACCAAGGTTACTATGGTATGGTGACAGGGGATATTTTCCCTTTGGATGCTAATTCTGTTGGGGATACTATCAACCGTGGA GGAACGTTTTTACGTTCAGCACGTTATCCTGAATTTGCTGAACTTGAAGGTCAGCTTAAAGGGATTGAACAGCTTAAAAAACACGGTATTGAAGGT GTAGTAGTTATCGGTGGTGATGGTTCTTATCATGGTGCTATGCGTCTAACTGAGCACGGTTTCCCAGCTGTTGGTTTGCCGGGTACAATTGATAAC GATATCGTTGGCACTGACTATACTATTGGTTTTGACACAGCAGTTGCGACAGCAGTTGAGAATCTTGACCGTCTTCGTGATACATCAGCAAGTCAT AACCGTACTTTTGTTGTTGAGGTTATGGGAAGAAATGCAGGAGATATCGCTCTTTGGTCAGGTATCGCTGCAGGTGCAGATCAAATTATTGTTCCT GAAGAAGAGTTCAATATTGATGAAGTTGTCTCAAATGTTAGAGCTGGCTATGCAGCTGGTAAACATCACCAAATCATCGTCCTTGCAGAAGGTGTT ATGAGTGGTGATGAGTTTGCAAAAACAATGAAAGCAGCAGGAGACGATAGCGATCTTCGTGTGACGAATTTAGGACATCTGCTCCGTGGTGGTAGT CCGACGGCTCGTGATCGTGTCTTAGCATCTCGTATGGGAGCGTACGCTGTTCAATTGTTGAAAGAAGGTCGTGGTGGTTTAGCCGTTGGTGTCCAC AACGAAGAAATGGTTGAAAGTCCAATTTTAGGTTTAGCAGAAGAAGGTGCTTTGTTCAGCTTGACTGATGAAGGAAAAATCGTTGTTAATAATCCG CATAAAGCGGACCTTCGCTTGGCAGCACTTAATCGTGACCTTGCCAACCAAAGTAGTAAA SEQ ID NO. 16 MKRIAVLTSGGDAPGMNAAIRAVVRKAISEGMEVYGINQGYYGMVTGDIFPLDANSVGDTINRGGTFLRSARYPEFAELEGQLKGIEQLKKHGIEG VVVIGGDGSYHGAMRLTEHGFPAVGLPGTIDNDIVGTDYTIGFDTAVATAVENLDRLRDTSASHHRTFVVEVMGRNAGDIALWSGIAAGADQIIVP EEEFNIDEVVSNVRAGYAAGKHHQIIVLAEGVMSGDEFAKTMKAAGDDSDLRVTNLGHLLRGGSPTARDRVLASRMGAYAVQLLKEGRGGLAVGVH NEEMVESPILGLAEEGALFSLTDEGKIVVNNPHKADLRLAALNRDLANQSSK

The nucleotide and amino acid sequences of GBS 322 in Ref. 3 are SEQ ID 8539 and SEQ ID 8540. These sequences are set forth below as SEQ ID NOS 17 and 18:

SEQ ID NO. 17 ATGAATAAAAAGGTACTATTGACATCGACAATGGCAGCTTCGCTATTATCAGTCGCAAGTGTTCAAGCACAAGAAACAGATACGACGTGGACAGCA CGTACTGTTTCAGAGGTAAAGGCTGATTTGGTAAAGCAAGACAATAAATCATCATATACTGTGAAATATGGTGATACACTAAGCGTTATTTCAGAA GCAATGTCAATTGATATGAATGTCTTAGCAAAAATAAATAACATTGCAGATATCAATCTTATTTATCCTGAGACAACACTGACAGTAACTTACGAT CAGAAGAGTCATACTGCCACTTCAATGAAAATAGAAACACCAGCAACAAATGCTGCTGGTCAAACAACAGCTACTGTGGATTTGAAAACCAATCAA GTTTCTGTTGCAGACCAAAAAGTTTCTCTCAATACAATTTCGGAAGGTATGACACCAGAAGCAGCAACAACGATTGTTTCGCCAATGAAGACATAT TCTTCTGCGCCAGCTTTGAAATCAAAAGAAGTATTAGCACAAGAGCAAGCTGTTAGTCAAGCAGCAGCTAATGAACAGGTATCACCAGCTCCTGTG AAGTCGATTACTTCAGAAGTTCCAGCAGCTAAAGAGGAAGTTAAACCAACTCAGACGTCAGTCAGTCAGTCAACAACAGTATCACCAGCTTCTGTT GCCGCTGAAACACCAGCTCCAGTAGCTAAAGTAGCACCGGTAAGAACTGTAGCAGCCCCTAGAGTGGCAAGTGTTAAAGTAGTCACTCCTAAAGTA GAAACTGGTGCATCACCAGAGCATGTATCAGCTCCAGCAGTTCCTGTGACTACGACTTCACCAGCTACAGACAGTAAGTTACAAGCGACTGAAGTT AAGAGCGTTCCGGTAGCACAAAAAGCTCCAACAGCAACACCGGTAGCACAACCAGCTTCAACAACAAATGCAGTAGCTGCACATCCTGAAAATGCA GGGCTCCAACCTCATGTTGCAGCTTATAAAGAAAAAGTAGCGTCAACTTATGGAGTTAATGAATTCAGTACATACCGTGCGGGAGATCCAGGTGAT CATGGTAAAGGTTTAGCAGTTGACTTTATTGTAGGTACTAATCAAGCACTTGGTAATAAAGTTGCACAGTACTCTACACAAAATATGGCAGCAAAT AACATTTCATATGTTATCTGGCAACAAAAGTTTTACTCAAATACAAACAGTATTTATGGACCTGCTAATACTTGGAATGCAATGCCAGATCGTGGT GGCGTTACTGCCAACCACTATGACCACGTTCACGTATCATTTAACAAATAATATAAAAAAGGAAGCTATTTGGCTTCTTTTTTATATGCCTTGAAT AGACTTTCAAGGTTCTTATATAATTTTTATTA SEQ ID NO. 18 MNKKVLLTSTMAASLLSVASVQAQETDTTWTARTVSEVKADLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNIADINLIYPETTLTVTYD QKSHTATSMKIETPATNAAGQTTATVDLKTNQVSVADQKVSLNTISEGMTPEAATTIVSPMKTYSSAPALKSKEVLAQEQAVSQAAANEQVSPAPV KSITSEVPAAKEEVKPTQTSVSQSTTVSPASVAAETPAPVAKVAPVRTVAAPRVASVKVVTPKVETGASPEHVSAPAVPVTTTSPATDSKLQATEV KSVPVAQKAPTATPVAQPASTTNAVAAHPENAGLQPHVAAYKEKVASTYGVNEFSTYRAGDPGDHGKGLAVDFIVGTNQALGNKVAQYSTQNMAAN NISYVIWQQKFYSNTNSIYGPANTWNAMPDRGGVTANHYDHVHVSFNK

The nucleotide and amino acid sequences of GBS 328 in Ref. 3 are SEQ ID 6015 and SEQ ID 6016. These sequences are set forth below as SEQ ID NOS 19 and 20:

SEQ ID NO. 19 ATGAAAAAGAAAATTATTTTGAAAAGTAGTGTTCTTGGTTTAGTCGCTGGGACTTCTATTATGTTCTCAAGCGTGTTCGCGGACCAAGTCGGTGTC CAAGTTATAGGCGTCAATGACTTTCATGGTGCACTTGACAATACTGGAACAGCAAATATGCCTGATGGAAAAGTTGCTAATGCTGGTACTGCTGCT CAATTAGATGCTTATATGGATGACGCTCAAAAAGATTTCAAACAAACTAACCCTAATGGTGAAAGCATTAGGGTTCAAGCAGGCGATATGGTTGGA GCAAGTCCAGCCAACTCTGGGCTTCTTCAAGATGAACCAACTGTCAAAAATTTTAATGCAATGAATGTTGAGTATGGCACATTGGGTAACCATGAA TTTGATGAAGGGTTGGCAGAATATAATCGTATCGTTACTGGTAAAGCCCCTGCTCCAGATTCTAATATTAATAATATTACGAAATCATACCCACAT GAAGCTGCAAAACAAGAAATTGTAGTGGCAAATGTTATTGATAAAGTTAACAAACAAATTCCTTACAATTGGAAGCCTTACGCTATTAAAAATATT CCTGTAAATAACAAAAGTGTGAACGTTGGCTTTATCGGGATTGTCACCAAAGACATCCCAAACCTTGTCTTACGTAAAAATTATGAACAATATGAA TTTTTAGATGAAGCTGAAACAATCGTTAAATACGCCAAAGAATTACAAGCTAAAAATGTCAAAGCTATTGTAGTTCTCGCACATGTACCTGCAACA AGTAAAAATGATATTGCTGAAGGTGAAGCAGCAGAAATGATGAAAAAAGTCAATCAACTCTTCCCTGAAAATAGCGTAGATATTGTCTTTGCTGGA CACAATCATCAATATACAAATGGTCTTGTTGGTAAAACTCGTATTGTACAAGCGCTCTCTCAAGGAAAAGCCTATGCTGATGTACGTGGTGTCTTA GATACTGATACACAAGATTTCATTGAGACCCCTTCAGCTAAAGTAATTGCAGTTGCTCCTGGTAAAAAAACAGGTAGTGCCGATATTCAAGCCATT GTTGACCAAGCTAATACTATCGTTAAACAAGTAACAGAAGCTAAAATTGGTACTGCCGAGGTAAGTGTCATGATTACGCGTTCTGTTGATCAAGAT AATGTTAGTCCGGTAGGCAGCCTCATCACAGAGGCTCAACTAGCAATTGCTCGAAAAAGCTGGCCAGATATCGATTTTGCCATGACAAATAATGGT GGCATTCGTGCTGACTTACTCATCAAACCAGATGGAACAATCACCTGGGGAGCTGCACAAGCAGTTCAACCTTTTGGTAATATCTTACAAGTCGTC GAAATTACTGGTAGAGATCTTTATAAAGCACTCAACGAACAATACGACCAAAAACAAAATTTCTTCCTTCAAATAGCTGGTCTGCGATACACTTAC ACAGATAATAAAGAGGGCGGGGAAGAAACACCATTTAAAGTTGTAAAAGCTTATAAATCAAATGGTGAGGAAATCAATCCTGATGCAAAATACAAA TTAGTTATCAATGACTTTTTATTCGGTGGTGGTGATGGCTTTGCAAGCTTCAGAAATGCCAAACTTCTAGGAGCCATTAACCCCGATACAGAGGTA TTTATGGCCTATATCACTGATTTAGAAAAAGCTGGTAAAAAAGTGAGCGTTCCAAATAATAAACCTAAAATCTATGTCACTATGAAGATGGTTAAT GAAACTATTACACAAAATGATGGTACACATAGCATTATTAAGAAACTTTATTTAGATCGACAAGGAAATATTGTAGCACAAGAGATTGTATCAGAC ACTTTAAACCAAACAAAATCAAAATCTACAAAAATCAACCCTGTAACTACAATTCACAAAAAACAATTACACCAATTTACAGCTATTAACCCTATG AGAAATTATGGCAAACCATCAAACTCCACTACTGTAAAATCAAAACAATTACCAAAAACAAACTCTGAATATGGACAATCATTCCTTATGTCTGTC TTTGGTGTTGGACTTATAGGAATTGCTTTAAATACAAAGAAAAAACATATGAAA SEQ ID NO. 20 MKKKIILKSSVLGLVAGTSIMFSSVFADQVGVQVIGVNDFHGALDNTGTANMPDGKVANAGTAAQLDAYMDDAQKDFKQTNPNGESIRVQAGDMVG ASPANSGLLQDEPTVKNFNAMNVEYGTLGNHEFDEGLAEYNRIVTGKAPAPDSNINNITKSYPHEAAKQEIVVANVIDKVNKQIPYNWKPYAIKNI PVNNKSVNVGFIGIVTKDIPNLVLRKNYEQYEFLDEAETIVKYAKELQAKNVKAIVVLAHVPATSKNDIAEGEAAEMMKKVNQLFPENSVDIVFAG HNHQYTNGLVGKTRIVQALSQGKAYADVRGVLDTDTQDFIETPSAKVIAVAPGKKTGSADIQAIVDQANTIVKQVTEAKIGTAEVSVMITRSVDQD NVSPVGSLITEAQLAIARKSWPDIDFAMTNNGGIRADLLIKPDGTITWGAAQAVQPFGNILQVVEITGRDLYKALNEQYDQKQNFFLQIAGLRYTY TDNKEGGEETPFKVVKAYKSNGEEINPDAKYKLVINDFLFGGGDGFASFRNAKLLGAINPDTEVFMAYITDLEKAGKKVSVPNNKPKIYVTMKMVN ETITQNDGTHSIIKKLYLDRQGNIVAQEIVSDTLNQTKSKSTKINPVTTIHKKQLHQFTAINPMRNYGKPSNSTTVKSKQLPKTNSEYGQSFLMSV FGVGLIGIALNTKKKHMK

The nucleotide and amino acid sequences of GBS 330 in Ref. 3 are SEQ ID 8791 and SEQ ID 8792. These sequences are set forth below as SEQ ID NOS 21 and 22:

SEQ ID NO. 21 ATGAATAAACGCGTAAAAATCGTTGCAACACTTGGTCCTGCGGTTGAATTCCGTGGTGGTAAGAAGTTTGGTGAGTCTGGATACTGGGGTGAAAGC CTTGACGTAGAAGCTTCAGCAGAAAAAATTGCTCAATTGATTAAAGAAGGTGCTAACGTTTTCCGTTTCAACTTCTCACATGGAGATCATGCTGAG CAAGGAGCTCGTATGGCTACTGTTCGTAAAGCAGAAGAGATTGCAGGACAAAAAGTTGGCTTCCTCCTTGATACTAAAGGACCTGAAATTCGTACA GAACTTTTTGAAGATGGTGCAGATTTCCATTCATATACAACAGGTACAAAATTACGTGTTGCTACTAAGCAAGGTATCAAATCAACTCCAGAAGTG ATTGCATTGAATGTTGCTGGTGGACTTGACATCTTTGATGACGTTGAAGTTGGTAAGCAAATCCTTGTTGATGATGGTAAACTAGGTCTTACTGTG TTTGCAAAAGATAAAGACACTCGTGAATTTGAAGTAGTTGTTGAGAATGATGGCCTTATTGGTAAACAAAAAGGTGTAAACATCCCTTATACTAAA ATTCCTTTCCCAGCACTTGCAGAACGCGATAATGCTGATATCCGTTTTGGACTTGAGCAAGGACTTAACTTTATTGCTATCTCATTTGTACGTACT GCTAAAGATGTTAATGAAGTTCGTGCTATTTGTGAAGAAACTGGSMATGGACACGTTAAGTTGTTTGCTAAAATTGAAAATCAACAAGGTATCGAT AATATTGATGAGATTATCGAAGCAGCAGATGGTATTATGATTGCTCGTGGTGATATGGGTATCGAAGTTCCATTTGAAATGGTTCCAGTTTACCAA AAAATGATCATTACTAAAGTTAATGCAGCTGGTAAAGCAGTTATTACAGCAACAAATATGCTTGAAACAATGACTGATAAACCACGTGCGACTCGT TCAGAAGTATCTGATGTCTTCAATGCTGTTATTGATGGTACTGATGCTACAATGCTTTCAGGTGAGTCAGCTAATGGTAAATACCCAGTTGAGTCA GTTCGTACAATGGCTACTATTGATAAAAATGCTCAAACATTACTCAATGAGTATGGTCGCTTAGACTCATCTGCATTCCCACGTAATAACAAAACT GATGTTATTGCATCTGCGGTTAAAGATGCAACACACTCAATGGATATCAAACTTGTTGTAACAATTACTGAAACAGGTAATACAGCTCGTGCCATT TCTAAATTCCGTCCAGATGCAGACATTTTGGCTGTTACATTTGATGAAAAAGTACAACGTTCATTGATGATTAACTGGGGTGTTATCCCTGTCCTT GCAGACAAACCAGCATCTACAGATGATATGTTTGAGGTTGCAGAACGTGTAGCACTTGAAGCAGGATTTGTTGAATCAGGCGATAATATCGTTATC GTTGCAGGTGTTCCTGTAGGTACAGGTGGAACTAACACAATGCGTGTTCGTACTGTTAAA SEQ ID NO. 22 MNKRVKIVATLGPAVEFRGGKKFGESGYWGESLDVEASAEKIAQLIKEGANVFRFNFSHGDHAEQGARMATVRKAEEIAGQKVGFLLDTKGPEIRT ELFEDGADFHSYTTGTKLRVATKQGIKSTPEVIALNVAGGLDIFDDVEVGKQILVDDGKLGLTVFAKDKDTREFEVVVENDGLIGKQKGVNIPYTK IPFPALAERDNADIRFGLEQGLNFIAISFVRTAKDVNEVRAICEETGXGHVKLFAKIENQQGIDNIDEIIEAADGIMIARGDMGIEVPFEMVPVYQ KMIITKVNAAGKAVITATNMLETMTDKPRATRSEVSDVFNAVIDGTDATMLSGESANGKYPVESVRTMATIDKNAQTLLNEYGRLDSSAFPRNNKT DVIASAVKDATHSMDIKLVVTITETGNTARAISKFRPDADILAVTFDEKVQRSLMINWGVIPVLADKPASTDDMFEVAERVALEAGFVESGDNIVI VAGVPVGTGGTNTMRVRTVK

The nucleotide and amino acid sequences of GBS 338 in Ref. 3 are SEQ ID 8637 and SEQ ID 8638. These sequences are set forth below as SEQ ID NOS 23 and 24:

SEQ ID NO. 23 TTGTCTGCTATAATAGACAAAAAGGTGGTGATATTTATGTATTTAGCATTAATCGGTGATATCATTAATTCAAAACAGATACTTGA ACGTGAAACTTTCCAACAGTCTTTTCAGCAACTAATGACCGAACTATCTGATGTATATGGTGAAGAGCTGATTTCTCCATTCACTA TTACAGCTGGTGATGAATTTCAAGCTTTATTGAAACCATCAAAAAAGGTATTTCAAATTATTGACCATATTCAACTAGCTCTAAAA CCTGTTAATGTAAGGTTCGGCCTCGGTACAGGAAACATTATAACATCCATCAATTCAAATGAAAGTATCGGTGCTGATGGTCCTGC CTACTGGCATGCTCGCTCAGCTATTAATCATATACATGATAAAAATGATTATGGAACAGTTCAAGTAGCTATTTGCCTTGATGATG AAGACCAAAACCTTGAATTAACACTAAATAGTCTCATTTCAGCTGGTGATTTTATCAAGTCAAAATGGACTACAAACCATTTTCAA ATGCTTGAGCACTTAATACTTCAAGATAATTATCAAGAACAATTTCAACATCAAAAGTTAGCCCAACTGGAAAATATTGAACCTAG TGCGCTGACTAAACGCCTTAAAGCAAGCGGTCTGAAGATTTACTTAAGAACGAGAACACAGGCAGCCGATCTATTAGTTAAAAGTT GCACTCAAACTAAAGGGGGAAGCTATGATTTC SEQ ID NO. 24 MSAIIDKKVVIFMYLALIGDIINSKQILERETFQQSFQQLMTELSDVYGEELISPFTITAGDEFQALLKPSKKVFQIIDHIQLALKPVNVRFGLGTG NIITSINSNESIGADGPAYWHARSAINHIHDKNDYGTVQVAICLDDEDQNLELTLNSLISAGDFIKSKWTTNHFQMLEHLILQDNYQEQFQHQKLAQ LENIEPSALTKRLKASGLKIYLRTRTQAADLLVKSCTQTKGGSYDF

The nucleotide and amino acid sequences of GBS 358 in Ref. 3 are SEQ ID 3183 and SEQ ID 3184. These sequences are set forth below as SEQ ID NOS 25 and 26:

SEQ ID NO. 25 ATGTTTTATACAATTGAAGAGCTGGTAGAGCAAGCTAATAGCCAACATAAGGGTAACATAGCAGAGCTCATGATCCAAACGGAAATTGAAATGACT GGTAGAAGTCGTGAAGAAATTCGTTATATTATGTCCCGAAATCTTGAAGTCATGAAAGCTTCTGTTATTGATGGATTAACCCCTAGTAAATCAATC AGTGGTTTAACAGGCGGTGATGCTGTCAAGATGGATCAATATTTACAATCAGGAAAAACTATTTCAGATACCACAATCCTAGCTGCCGTTAGGAAT GCTATGGCTGTTAATGAGTTAAATGCTAAGATGGGACTGGTCTGTGCAACACCAACTGCAGGTAGTGCAGGATGTTTACCAGCTGTGATTTCTACA GCCATTGAAAAGCTTAATTTAACAGAAGAAGAGCAACTTGATTTTCTATTTACAGCCGGCGCATTTGGTCTCGTCATTGGTAATAATGCCTCTATC TCAGGTGCAGAAGGAGGTTGCCAAGCTGAAGTTGGGTCAGCTAGTGCTATGGCTGCGGCTGCTTTAGTTATGGCTGCTGGAGGTACTCCTTTCCAA GCTAGCCAAGCTATAGCATTTGTTATTAAAAATATGCTTGGACTTATCTGTGACCCTGTTGCAGGTTTAGTTGAAGTCCCTTGTGTGAAGCGGAAT GCTCTTGGATCAAGTTTTGCACTTGTTGCTGCTGATATGGCCTTGGCTGGTATTGAATCGCAAATTCCAGTAGATGAAGTTATTGATGCAATGTAT CAAGTTGGATCAAGTTTACCGACTGCTTTTCGTGAGACTGCAGAAGGAGGACTTGCTGCCACGCCGACAGGAAGACGTTATAGTAAAGAAATTTTT GGGGAA SEQ ID NO. 26 MFYTIEELVEQANSQHKGNIAELMIQTEIEMTGRSREEIRYIMSRNLEVMKASVIDGLTPSKSISGLTGGDAVKMDQYLQSGKTISDTTILAAVRN AMAVNELNAKMGLVCATPTAGSAGCLPAVISTAIEKLNLTEEEQLDFLFTAGAFGLVIGNNASISGAEGGCQAEVGSASAMAAAALVMAAGGTPFQ ASQAIAFVIKNMLGLICDPVAGLVEVPCVKRNALGSSFALVAADMALAGIESQIPVDEVIDAMYQVGSSLPTAFRETAEGGLAATPTGRRYSKEIF GE

The nucleotide and amino acid sequences of GBS 361 in Ref. 3 are SEQ ID 8769 and SEQ ID 8770. These sequences are set forth below as SEQ ID NOS 27 and 28:

SEQ ID NO. 27 ATGAGCGTATATGTTAGTGGAATAGGAATTATTTCTTCTTTGGGAAAGAATTATAGCGAGCATAAACAGCATCTCTTCGACTTAAAAGAAGGAATTT CTAAACATTTATATAAAAATCACGACTCTATTTTAGAATCTTATACAGGAAGCATAACTAGTGACCCAGAGGTTCCTGAGCAATACAAAGATGAGAC ACGTAATTTTAAATTTGCTTTTACCGCTTTTGAAGAGGCTCTTGCTTCTTCAGGTGTTAATTTAAAAGCTTATCATAATATTGCTGTGTGTTTAGGG ACCTCACTTGGGGGAAAGAGTGCTGGTCAAAATGCCTTGTATCAATTTGAAGAAGGAGAGCGTCAAGTAGATGCTAGTTTATTAGAAAAAGCATCTG TTTACCATATTGCTGATGAATTGATGGCTTATCATGATATTGTGGGAGCTTCGTATGTTATTTCAACCGCCTGTTCTGCAAGTAATAATGCCGTAAT ATTAGGAACACAATTACTTCAAGATGGCGATTGTGATTTAGCTATTTGTGGTGGCTGTGATGAGTTAAGTGATATTTCTTTAGCAGGCTTCACATCA CTAGGAGCTATTAATACAGAAATGGCATGTCAGCCCTATTCTTCTGGAAAAGGAATCAATTTGGGTGAGGGCGCTGGTTTTGTTGTTCTTGTCAAAG ATCAGTCCTTAGCTAAATATGGAAAAATTATCGGTGGTCTTATTACTTCAGATGGTTATCATATAACAGCACCTAAGCCAACAGGTGAAGGGGCGGC ACAGATTGCAAAGCAGCTAGTGACTCAAGCAGGTATTGACTACAGTGAGATTGACTATATTAACGGTCACGGTACAGGTACTCAAGCTAATGATAAA ATGGAAAAAAATATGTATGGTAAGTTTTTCCCGACAACGACATTGATCAGCAGTACCAAGGGGCAAACGGGTCATACTCTAGGGGCTGCAGGTATTA TCGAATTGATTAATTGTTTAGCGGCAATAGAGGAACAGACTGTACCAGCAACTAAAAATGAGATTGGGATAGAAGGTTTTCCAGAAAATTTTGTCTA TCATCAAAAGAGAGAATACCCAATAAGAAATGCTTTAAATTTTTCGTTTGCTTTTGGTGGAAATAATAGTGGTGTCTTATTGTCATCTTTAGATTCA CCTCTAGAAACATTACCTGCTAGAGAAAATCTTAAAATGGCTATCTTATCATCTGTTGCTTCCATTTCTAAGAATGAATCACTTTCTATAACCTATG AAAAAGTTGCTAGTAATTTCAACGACTTTGAAGCATTACGCTTTAAAGGGGCTAGACCACCCAAAACTGTCAACCCAGCACAATTTAGGAAAATGGA TGATTTTTCCAAAATGGTTGCCGTAACAACAGCTCAAGCACTAATAGAAAGCAATATTAATCTAAAAAAACAAGATACTTCAAAAGTAGGAATTGTA TTTACAACACTTTCTGGACCAGTTGAGGTTGTTGAAGGTATTGAAAAGCAAATCACAACAGAAGGATATGCACATGTTTCTGCTTCACGATTCCCGT TTACAGTAATGAATGCAGCAGCTGGTATGCTTTCTATCATTTTTAAAATAACAGGTCCTTTATCTGTCATTTCGACAAATAGTGGAGCGCTTGATGG TATACAATATGCCAAGGAAATGATGCGTAACGATAATCTAGACTATGTGATTCTTGTTTCTGCTAATCAGTGGACAGACATGAGTTTTATGTGGTGG CAACAATTAAACTATGATAGTCAAATGTTTGTCGGTTCTGATTATTGTTCAGCACAAGTCCTCTCTCGTCAAGCATTGGATAATTCTCCTATAATAT TAGGTAGTAAACAATTAAAATATAGCCATAAAACATTCACAGATGTGATGACTATTTTTGATGCTGCGCTTCAAAATTTATTATCAGACTTAGGACT AACCATAAAAGATATCAAAGGTTTCGTTTGGAATGAGCGGAAGAAGGCAGTTAGTTCAGATTATGATTTCTTAGCGAACTTGTCTGAGTATTATAAT ATGCCAAACCTTGCTTCTGGTCAGTTTGGATTTTCATCTAATGGTGCTGGTGAAGAACTGGACTATACTGTTAATGAAAGTATAGAAAAGGGCTATT ATTTAGTCCTATCTTATTCGATCTTCGGTGGTATCTCTTTTGCTATTATTGAAAAAAGG SEQ ID NO. 28 MSVYVSGIGIISSLGKNYSEHKQHLFDLKEGISKHLYKNHDSILESYTGSITSDPEVPEQYKDETRNFKFAFTAFEEALASSGVNLKAYHNIAVCLG TSLGGKSAGQNALYQFEEGERQVDASLLEKASVYHIADELMAYHDIVGASYVISTACSASNNAVILGTQLLQDGDCDLAICGGCDELSDISLAGFTS LGAINTEMACQPYSSGKGINLGEGAGFVVLVKDQSLAKYGKIIGGLITSDGYHITAPKPTGEGAAQIAKQLVTQAGIDYSEIDYINGHGTGTQANDK MEKNMYGKFFPTTTLISSTKGQTGHTLGAAGIIELINCLAAIEEQTVPATKNEIGIEGFPENFVYHQKREYPIRNALNFSFAFGGNNSGVLLSSLDS PLETLPARENLKMAILSSVASISKNESLSITYEKVASNFNDFEALRFKGARPPKTVNPAQFRKMDDFSKMVAVTTAQALIESNINLKKQDTSKVGIV FTTLSGPVEVVEGIEKQITTEGYAHVSASRFPFTVMNAAAGMLSIIFKITGPLSVISTNSGALDGIQYAKEMMRNDNLDYVILVSANQWTDMSFMWW QQLNYDSQMFVGSDYCSAQVLSRQALDNSPIILGSKQLKYSHKTFTDVMTIFDAALQNLLSDLGLTIKDIKGFVWNERKKAVSSDYDFLANLSEYYN MPNLASGQFGFSSNGAGEELDYTVNESIEKGYYLVLSYSIEGGISFAIIEKR

The nucleotide and amino acid sequences of GBS 404 in Ref. 3 are SEQ ID 8799 and SEQ ID 8800. These sequences are set forth below as SEQ ID NOS 29 and 30:

SEQ ID NO. 29 ATGAAAATAGATGACCTAAGAAAAAGCGACAATGTTGAAGATCGTCGCTCCAGTAGCGGAGGTTCATTCTCTAGCGGAGGAAGTGGATTACCGATT CTTCAACTTTTATTGCTGCGAGGGAGTTGGAAAACCAAGCTTGTGGTTTTAATCATCTTACTGCTACTTGGCGGAGGGGGACTAACCAGCATTTTT AATGACTCATCCTCACCTTCTAGTTACCAATCTCAGAATGTCTCACGTTCTGTTGATAATAGCGCAACGAGAGAACAAATCGATTTCGTTAATAAA GTCCTTGGCTCAACTGAGGATTTCTGGTCACAAGAATTCCAAACCCAAGGTTTTGGAAATTATAAGGAACCAAAACTTGTTCTTTACACCAATTCA ATTCAAACAGGTTGTGGTATAGGTGAATCTGCTTCAGGACCATTTTATTGTTCAGCAGATAAAAAAATCTATCTTGATATTTCTTTTTACAATGAA TTATCACATAAATATGGTGCTACTGGTGATTTTGCTATGGCCTACGTCATCGCCCACGAAGTTGGTCACCACATTCAAACAGAGTTAGGCATTATG GATAAGTATAATAGAATGCGACACGGACTTACTAAGAAAGAAGCAAATGCTTTAAATGTTCGGCTAGAACTTCAAGCAGATTATTATGCAGGGGTA TGGGCTCACTACATCAGGGGAAAAAATCTCTTAGAACAAGGAGACTTTGAAGAGGCCATGAATGCTGCCCACGCCGTCGGAGACGATACCCTTCAG AAAGAAACCTACGGAAAATTAGTGCCTGATAGCTTTACCCATGGAACAGCTGAACAACGCCAACGTTGGTTTAACAAAGGCTTTCAATATGGTGAC ATCCAACACGGTGATACTTTCTCCGTAGAACATCTA SEQ ID NO. 30 MKIDDLRKSDNVEDRRSSSGGSFSSGGSGLPILQLLLLRGSWKTKLVVLIILLLLGGGGLTSIFNDSSSPSSYQSQNVSRSVDNSATREQIDFVNK VLGSTEDFWSQEFQTQGFGNYKEPKLVLYTNSIQTGCGIGESASGPFYCSADKKIYLDISFYNELSHKYGATGDFAMAYVIAHEVGHHIQTELGIM DKYNRMRHGLTKKEANALNVRLELQADYYAGVWAHYIRGKNLLEQGDFEEAMNAAHAVGDDTLQKETYGKLVPDSFTHGTAEQRQRWFNKGFQYGD IQHGDTFSVEHL

The nucleotide and amino acid sequences of GBS 656 in Ref. 3 are SEQ ID 9323 and SEQ ID 9324. These sequences are set forth below as SEQ ID NOS 31 and 32:

SEQ ID NO. 31 ATGAAAAGATTACATAAACTGTTTATAACCGTAATTGCTACATTAGGTATGTTGGGGGTAATGACCTTTGGTCTTCCAACGCAGCCGCAAAACGTA ACGCCGATAGTACATGCTGATGTCAATTCATCTGTTGATACGAGCCAGGAATTTCAAAATAATTTAAAAAATGCTATTGGTAACCTACCATTTCAA TATGTTAATGGTATTTATGAATTAAATAATAATCAGACAAATTTAAATGCTGATGTCAATGTTAAAGCGTATGTTCAAAATACAATTGACAATCAA CAAAGACTATCAACTGCTAATGCAATGCTTGATAGAACCATTCGTCAATATCAAAATCGCAGAGATACCACTCTTCCCGATGCAAATTGGAAACCA TTAGGTTGGCATCAAGTAGCTACTAATGACCATTATGGACATGCAGTCGACAAGGGGCATTTAATTGCCTATGCTTTAGCTGGAAATTTCAAAGGT TGGGATGCTTCCGTGTCAAATCCTCAAAATGTTGTCACACAAACAGCTCATTCCAACCAATCAAATCAAAAAATCAATCGTGGACAAAATTATTAT GAAAGCTTAGTTCGTAAGGCGGTTGACCAAAACAAACGTGTTCGTTACCGTGTAACTCCATTGTACCGTAATGATACTGATTTAGTTCCATTTGCA ATGCACCTAGAAGCTAAATCACAAGATGGCACATTAGAATTTAATGTTGCTATTCCAAACACACAAGCATCATACACTATGGATTATGCAACAGGA GAAATAACACTAAAT SEQ ID NO. 32 MKRLHKLFITVIATLGMLGVMTFGLPTQPQNVTPIVHADVNSSVDTSQEFQNNLKNAIGNLPFQYVNGIYELNNNQTNLNADVNVKAYVQNTIDNQ QRLSTANAMLDRTIRQYQNRRDTTLPDANWKPLGWHQVATNDHYGHAVDKGHLTAYALAGNFKGWDASVSNPQNVVTQTAHSNQSNQKINRGQNYY ESLVRKAVDQNKRVRYRVTPLYRNDTDLVPFAMHLEAKSQDGTLEFNVAIPNTQASYTMDYATGEITLN

The nucleotide and amino acid sequences of GBS 690 in Ref. 3 are SEQ ID 9965 and SEQ ID 9966. These sequences are set forth as SEQ ID NOS 33 and 34 below:

SEQ ID NO. 33 ATGAGTAAACGACAAAATTTAGGAATTAGTAAAAAAGGAGCAATTATATCAGGGCTCTCAGTGGCACTAATTGTAGTAATAGGTGGCTTTTTATGG GTACAATCTCAACCTAATAAGAGTGCAGTAAAAACTAACTACAAAGTTTTTAATGTTAGAGAAGGAAGTGTTTCGTCCTCAACTCTTTTGACAGGA AAAGCTAAGGCTAATCAAGAACAGTATGTGTATTTTGATGCTAATAAAGGTAATCGAGCAACTGTCACAGTTAAAGTGGGTGATAAAATCACAGCT GGTCAGCAGTTAGTTCAATATGATACAACAACTGCACAAGCAGCCTACGACACTGCTAATCGTCAATTAAATAAAGTAGCGCGTCAGATTAATAAT CTAAAGACAACAGGAAGTCTTCCAGCTATGGAATCAAGTGATCAATCTTCTTCATCATCACAAGGACAAGGGACTCAATCGACTAGTGGTGCGACG AATCGTCTACAGCAAAATTATCAAAGTCAAGCTAATGCTTCATACAACCAACAACTTCAAGATTTGAATGATGCTTATGCAGATGCACAGGCAGAA GTAAATAAAGCACAAAAAGCATTGAATGATACTGTTATTACAAGTGACGTATCAGGGACAGTTGTTGAAGTTAATAGTGATATTGATCCAGCTTCA AAAACTAGTCAAGTACTTGTCCATGTAGCAACTGAAGGTAAACTCCAAGTACAAGGAACGATGAGTGAGTATGATTTGGCTAATGTTAAAAAAGAC CAGGCTGTTAAAATAAAATCTAAGGTCTATCCTGACAAGGAATGGGAAGGTAAAATTTCATATATCTCAAATTATCCAGAAGCAGAAGCAAACAAC AATGACTCTAATAACGGCTCTAGTGCTGTAAATTATAAATATAAAGTAGATATTACTAGCCCTCTCGATGCATTAAAACAAGGTTTTACCGTATCA GTTGAAGTAGTTAATGGAGATAAGCACCTTATTGTCCCTACAAGTTCTGTGATAAACAAAGATAATAAACACTTTGTTTGGGTATACAATGATTCT AATCGTAAAATTTCCAAAGTTGAAGTCAAAATTGGTAAAGCTGATGCTAAGACACAAGAAATTTTATCAGGTTTGAAAGCAGGACAAATCGTGGTT ACTAATCCAAGTAAAACCTTCAAGGATGGGCAAAAAATTGATAATATTGAATCAATCGATCTTAACTCTAATAAGAAATCAGAGGTGAAA SEQ ID NO. 34 MSKRQNLGISKKGAIISGLSVALIVVIGGFLWVQSQPNKSAVKTNYKVFNVREGSVSSSTLLTGKAKANQEQYVYFDANKGNRATVTVKVGDKITAG QQLVQYDTTTAQAAYDTANRQLNKVARQINNLKTTGSLPAMESSDQSSSSSQGQGTQSTSGATNRLQQNYQSQANASYNQQLQDLNDAYADAQAEVN KAQKALNDTVITSDVSGTVVEVNSDIDPASKTSQVLVEVATEGKLQVQGTMSEYDLANVKKDQAVKIKSKVYPDKEWEGKISYISNYPEAEANNNDS NNGSSAVNYKYKVDITSPLDALKQGFTVSVEVVNGDKHLIVPTSSVINKDNKHFVWVYNDSNRKISKVEVKIGKADAKTQEILSGLKAGQIVVTNPS KTFKDGQKIDNIESIDLNSNKKSEVK

The nucleotide and amino acid sequences of GBS 691 in Ref. 3 are SEQ ID 3691 and SEQ ID 3692. These sequences are set forth as SEQ ID NOS 35 and 36 below:

SEQ ID NO. 35 ATGAAAAAAATTGGAATTATTGTCCTCACACTACTGACCTTCTTTTTGGTATCTTGCGGACAACAAACTAAACAAGAAAGCACTAAAACAACTATT TCTAAAATGCCTAAAATTGAAGGCTTCACCTATTATGGAAAAATTCCTGAAAATCCGAAAAAAGTAATTAATTTTACATATTCTTACACTGGGTAT TTATTAAAACTAGGTGTTAATGTTTCAAGTTACAGTTTAGACTTAGAAAAAGATAGCCCCGTTTTTGGTAAACAACTGAAAGAAGCTAAAAAATTA ACTGCTGATGATACAGAAGCTATTGCCGCACAAAAACCTGATTTAATCATGGTTTTCGATCAAGATCCAAACATCAATACTCTGAAAAAAATTGCA CCAACTTTAGTTATTAAATATGGTGCACAAAATTATTTAGATATGATGCCAGCCTTGGGGAAAGTATTCGGTAAAGAAAAAGAAGCTAATCAGTGG GTTAGCCAATGGAAAACTAAAACTCTCGCTGTCAAAAAAGATTTACACCATATCTTAAAGCCTAACACTACTTTTACTATTATGGATTTTTATGAT AAAAATATCTATTTATATGGTAATAATTTTGGACGCGGTGGAGAACTAATCTATGATTCACTAGGTTATGCTGCCCCAGAAAAAGTCAAAAAAGAT GTCTTTAAAAAAGGGTGGTTTACCGTTTCGCAAGAAGCAATCGGTGATTACGTTGGAGATTATGCCCTTGTTAATATAAACAAAACGACTAAAAAA GCAGCTTCATCACTTAAAGAAAGTGATGTCTGGAAGAATTTACCAGCTGTCAAAAAAGGGCACATCATAGAAAGTAACTACGACGTGTTTTATTTC TCTGACCCTCTATCTTTAGAAGCTCAATTAAAATCATTTACAAAGGCTATCAAAGAAAATACAAAT SEQ ID NO. 36 MKKIGIIVLTLLTFFLVSCGQQTKQESTKTTISKMPKIEGFTYYGKIPENPKKVINFTYSYTGYLLKLGVNVSSYSLDLEKDSPVF GKQLKEAKKLTADDTEAIAAQKPDLIMVFDQDPNINTLKKIAPTLVIKYGAQNYLDMMPALGKVFGKEKEANQWVSQWKTKTLAVK KDLHHILKPNTTFTIMDFYDKNIYLYGNNFGRGGELIYDSLGYAAPEKVKKDVFKKGWFTVSQEAIGDYVGDYALVNINKTTKKAA SSLKESDVWKNLPAVKKGHIIESNYDVFYFSDPLSLEAQLKSFTKAIKENTN

Other preferred polypeptide antigens include: GBS4 (SEQ ID 2 from Ref. 3); GBS22 (SEQ ID 8584 from Ref. 3); and GBS85 (SEQ ID 216 from Ref. 3), including polypeptides having amino acid sequences with sequence identity thereto etc.

The polypeptide is preferably not a C protein (alpha or beta or epsilon) or a R protein (Rib).

The nucleotide and amino acid sequences of GBS 4 in Ref. 3 are SEQ ID 1 and SEQ ID 2. These sequences are set forth below as SEQ ID NOS 37 and 38:

SEQ ID NO. 37 ATGAAAGTGAAAAATAAGATTTTAACGATGGTAGCACTTACTGTCTTAACATGTGCTACTTATTCATCAATCGGTTATGCTGATACAAGTGATAAGA ATACTGACACGAGTGTCGTGACTACGACCTTATCTGAGGAGAAAAGATCAGATGAACTAGACCAGTCTAGTACTGGTTCTTCTTCTGAAAATGAATC GAGTTCATCAAGTGAACCAGAAACAAATCCGTCAACTAATCCACCTACAACAGAACCATCGCAACCCTCACCTAGTGAAGAGAACAAGCCTGATGGT AGAACGAAGACAGAAATTGGCAATAATAAGGATATTTCTAGTGGAACAAAAGTATTAATTTCAGAAGATAGTATTAAGAATTTTAGTAAAGCAAGTA GTGATCAAGAAGAAGTGGATCGCGATGAATCATCATCTTCAAAAGCAAATGATGGGAAAAAAGGCCACAGTAAGCCTAAAAAGGAACTTCCTAAAAC AGGAGATAGCCACTCAGATACTGTAATAGCATCTACGGGAGGGATTATTCTGTTATCATTAAGTTTTTACAATAAGAAAATGAAACTTTAT SEQ ID NO. 38 MKVKNKILTMVALTVLTCATYSSIGYADTSDKNTDTSVVTTTLSEEKRSDELDQSSTGSSSENESSSSSEPETNPSTNPPTTEPSQPSPSEENKPDG RTKTEIGNNKDISSGTKVLISEDSIKNFSKASSDQEEVDRDESSSSKANDGKKGHSKPKKELPKTGDSHSDTVIASTGGIILLSLSFYNKKMKLY

The nucleotide and amino acid sequences of GBS 22 in Ref. 3 are SEQ 8583 and SEQ ID 8584. These sequences are set forth below as SEQ ID NOS 39 and 40:

SEQ ID NO. 39 ATGAAAAGGATACGGAAAAGCCTTATTTTTGTTCTCGGAGTAGTTACCCTAATTTGCTTATGTGCTTGTACTAAACAAAGCCAGCAAAAAAATGGCT TGTCAGTAGTGACTAGCTTTTATCCAGTATATTCCATTACAAAAGCAGTTTCTGGTGATTTGAATGATATTAAAATGATTCGATCACAGTCAGGTAT TCATGGTTTTGAACCCTCATCAAGTGATGTTGCTGCCATTTATGATGCTGATCTATTTCTTTATCATTCGCACACACTAGAAGCTTGGGCGAGACGT TTGGAACCTAGTTTGCATCACTCTAAAGTATCTGTAATTGAAGCTTCAAAAGGTATGACTTTGGATAAAGTTCATGGCTTAGAAGATGTAGAGGCAG AAAAAGGAGTAGATGAGTCAACCTTGTATGACCCTCACACTTGGAATGACCCTGTAAAAGTATCTGAGGAAGCACAACTCATCGCTACACAATTAGC TAAAAAGGATCCTAAAAACGCTAAGGTTTATCAAAAAAATGCTGATCAATTTAGTGACAAGGCAATGGCTATTGCAGAGAAGTATAAGCCAAAATTT AAAGCTGCAAAGTCTAAATACTTTGTGACTTCACATACAGCATTCTCATACTTAGCTAAGCGATACGGATTGACTCAGTTAGGTATTGCAGGTGTCT CAACCGAGCAAGAACCTAGTGCTAAAAAATTAGCCGAAATTCAGGAGTTTGTGAAAACATATAAGGTTAAGACTATTTTTGTTGAAGAAGGAGTCTC ACCTAAATTAGCTCAAGCAGTAGCTTCAGCTACTCGAGTTAAAATTGCAAGTTTAAGTCCTTTARAAGCAGTTCCCAAAAACAATAAAGATTACTTA GAAAATTTGGAAACTAATCTTAAGGTACTTGTCAAATCGTTAAATCAATAG SEQ ID NO. 40 MKRIRKSLIFVLGVVTLICLCACTKQSQQKNGLSVVTSFYPVYSITKAVSGDLNDIKMIRSQSGIHGFEPSSSDVAAIYDADLFLYHSHTLEAWARR LEPSLHHSKVSVIEASKGMTLDKVHGLEDVEAEKGVDESTLYDPHTWNDPVKVSEEAQLIATQLAKKDPKNAKVYQKNADQFSDKAMAIAEKYKPKF KAAKSKYFVTSHTAFSYLAKRYGLTQLGIAGVSTEQEPSAKKLAEIQEFVKTYKVKTIFVEEGVSPKLAQAVASATRVKIASLSPLXAVPKNNKDYL ENLETNLKVLVKSLNQ

The nucleotide and amino acid sequences of GBS 85 in Ref. 3 are SEQ ID 215 and SEQ ID 216. These sequences are set forth below as SEQ ID NOS 41 and 42:

SEQ ID NO. 41 ATGCCTAAGAAGAAATCAGATACCCCAGAAAAAGAAGAAGTTGTCTTAACGGAATGGCAAAAGCGTAACCTTGAATTTTTAAAAAAACGCAAAGAAG ATGAAGAAGAACAAAAACGTATTAACGAAAAATTACGCTTAGATAAAAGAAGTAAATTAAATATTTCTTCTCCTGAAGAACCTCAAAATACTACTAA AATTAAGAAGCTTCATTTTCCAAAGATTTCAAGACCTAAGATTGAAAAGAAACAGAAAAAAGAAAAAATAGTCAACAGCTTAGCCAAAACTAATCGC ATTAGAACTGCACCTATATTTGTAGTAGCATTCCTAGTCATTTTAGTTTCCGTTTTCCTACTAACTCCTTTTAGTAAGCAAAAAACAATAACAGTTA GTGGAAATCAGCATACACCTGATGATATTTTGATAGAGAAAACGAATATTCAAAAAAACGATTATTTCTTTTCTTTAATTTTTAAACATAAAGCTAT TGAACAACGTTTAGCTGCAGAAGATGTATGGGTAAAAACAGCTCAGATGACTTATCAATTTCCCAATAAGTTTCATATTCAAGTTCAAGAAAATAAG ATTATTGCATATGCACATACAAAGCAAGGATATCAACCTGTCTTGGAAACTGGAAAAAAGGCTGATCCTGTAAATAGTTCAGAGCTACCAAAGCACT TCTTAACAATTAACCTTGATAAGGAAGATAGTATTAAGCTATTAATTAAAGATTTAAAGGCTTTAGACCCTGATTTAATAAGTGAGATTCAGGTGAT AAGTTTAGCTGATTCTAAAACGACACCTGACCTCCTGCTGTTAGATATGCACGATGGAAATAGTATTAGAATACCATTATCTAAATTTAAAGAAAGA CTTCCTTTTTACAAACAAATTAAGAAGAACCTTAAGGAACCTTCTATTGTTGATATGGAAGTGGGAGTTTACACAACAACAAATACCATTGAATCAA CCCCTGTTAAAGCAGAAGATACAAAAAATAAATCAACTGATAAAACACAAACACAAAATGGTCAGGTTGCGGAAAATAGTCAAGGACAAACAAATAA CTCAAATACTAATCAACAAGGACAACAGATAGCAACAGAGCAGGCACCTAACCCTCAAAATGTTAAT SEQ ID NO. 42 MPKKKSDTPEKEEVVLTEWQKRNLEFLKKRKEDEEEQKRINEKLRLDKRSKLNISSPEEPQNTTKIKKLHFPKISRPKIEKKQKKEKIVNSLAKTNR IRTAPIFVVAFLVILVSVFLLTPFSKQKTITVSGNQHTPDDILIEKTNIQKNDYFFSLIFKHKAIEQRLAAEDVWVKTAQMTYQFPNKFHIQVQENK IIAYAHTKQGYQPVLETGKKADPVNSSELPKHFLTINLDKEDSIKLLIKDLKALDPDLISEIQVISLADSKTTPDLLLLDMHDGNSIRIPLSKFKER LPFYKQIKKNLKEPSIVDMEVGVYTTTNTIESTPVKAEDTKNKSTDKTQTQNGQVAENSQGQTNNSNTNQQGQQIATEQAPNPQNVN

GBS polypeptides of the invention may be present in the composition as individual separate polypeptides. It is preferred, however, that two or more (i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) of the antigens are expressed as a single polypeptide chain (a ‘hybrid’ polypeptide). Hybrid polypeptides offer two principal advantages: first, a polypeptide that may be unstable or poorly expressed on its own can be assisted by adding a suitable hybrid partner that overcomes the problem; second, commercial manufacture is simplified as only one expression and purification need be employed in order to produce two polypeptides which are both antigenically useful.

The hybrid polypeptide may comprise two or more polypeptide sequences from the first antigen group. Accordingly, the invention includes a composition comprising a first amino acid sequence and a second amino acid sequence, wherein said first and second amino acid sequences are selected from a GBS antigen or a fragment thereof. Preferably, the first and second amino acid sequences in the hybrid polypeptide comprise different epitopes.

The hybrid polypeptide may comprise one or more polypeptide sequences from different GBS serotypes. Accordingly, the invention includes a composition comprising a first amino acid sequence and a second amino acid sequence, said first amino acid sequence and said second amino acid sequence selected from a GBS serotype selected from the group consisting of serotypes Ia, Ib, Ia/c, II, III, IV, V, VI, VII and VIII. The first and second amino acid sequence may be from the same GBS serotype or they may be from different GBS serotypes. Preferably, the first and second amino acid sequence are selected a GBS serotype selected from the group consisting of serotypes II and V. Most preferably, at least one of the first and second amino acid sequences is from GBS serotype V. Preferably, the first and second amino acid sequences in the hybrid polypeptide comprise difference epitopes.

In one embodiment, the hybrid polypeptide comprises one or more GBS antigens from serotype V. Preferably, the hybrid polypeptide comprises a first amino acid sequence and a second amino acid sequence, said first amino acid sequence and said second amino acid sequence comprising a GBS antigen or a fragment thereof selected from the group consisting of GBS 80, GBS 91, GBS 104, GBS 147, GBS 173, GBS 276, GBS 305, GBS 313, GBS 322, GBS 328, GBS 330, GBS 338, GBS 358, GBS 361, GBS 404, GBS 656, GBS 690, and GBS 691. Preferably, the GBS antigen or fragment thereof is selected from the group consisting of GBS 80 and GBS 691. Preferably, the first and second amino acid sequences in the hybrid polypeptide comprise difference epitopes.

Hybrids consisting of amino acid sequences from two, three, four, five, six, seven, eight, nine, or ten GBS antigens are preferred. In particular, hybrids consisting of amino acid sequences from two, three, four, or five GBS antigens are preferred.

Different hybrid polypeptides may be mixed together in a single formulation. Within such combinations, a GBS antigen may be present in more than one hybrid polypeptide and/or as a non-hybrid polypeptide. It is preferred, however, that an antigen is present either as a hybrid or as a non-hybrid, but not as both.

Preferably, the GBS antigen in one of the hybrid polypeptides is GBS 80 or a fragment thereof. Accordingly, examples of two-antigen hybrids for use in the invention may comprise: (1) GBS 80 and GBS 91, (2) GBS 80 and GBS 104, (3) GBS 80 and GBS 147, (4) GBS 80 and GBS 173, (5) GBS 80 and GBS 276, (6) GBS 80 and GBS 305, (7) GBS 80 and GBS 313, (8) GBS 80 and GBS 322, (9) GBS 80 and GBS 328, (10) GBS 80 and GBS 330, (11) GBS 80 and GBS 338, (12) GBS 80 and GBS 358, (13) GBS 80 and GBS 361, (14) GBS 80 and GBS 404, (14) GBS 80 and GBS 404, (15) GBS 80 and GBS 656, (16) GBS 80 and GBS 690, and (17) GBS 80 and GBS 691. Preferably, a two-antigen hybrid for use in the invention comprises GBS 80 and GBS 691.

Hybrid polypeptides can be represented by the formula NH₂-A-{-X-L-}_(n)-B-COOH, wherein: X is an amino acid sequence of a GBS antigen or a fragment thereof; L is an optional linker amino acid sequence; A is an optional N-terminal amino acid sequence; B is an optional C-terminal amino acid sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

If a —X— moiety has a leader peptide sequence in its wild-type form, this may be included or omitted in the hybrid protein. In some embodiments, the leader peptides will be deleted except for that of the —X— moiety located at the N-terminus of the hybrid protein i.e. the leader peptide of X₁ will be retained, but the leader peptides of X₂ . . . X_(n) will be omitted. This is equivalent to deleting all leader peptides and using the leader peptide of X₁ as moiety -A-.

For each n instances of {—X-L-}, linker amino acid sequence -L- may be present or absent. For instance, when -n=2 the hybrid may be NH₂—X₁-L₁-X₂-L₂-COOH, NH₂—X₁-X₂—COOH, NH₂—X₁-L₁-X₂—COOH, NH₂—X₁-X₂-L₂-COOH, etc. Linker amino acid sequence(s) -L- will typically be short (e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise short peptide sequences which facilitate cloning, poly-glycine linkers (i.e. comprising Gly_(n) where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (i.e. His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be apparent to those skilled in the art. A useful linker is GSGGGG (SEQ ID 1), with the Gly-Ser dipeptide being formed from a BamHI restriction site, thus aiding cloning and manipulation, and the (Gly)₄ tetrapeptide being a typical poly-glycine linker.

-A- is an optional N-terminal amino acid sequence. This will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leader sequences to direct protein trafficking, or short peptide sequences which facilitate cloning or purification (e.g. histidine tags i.e. His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art. If X₁ lacks its own N-terminus methionine, -A- is preferably an oligopeptide (e.g. with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) which provides a N-terminus methionine.

-B- is an optional C-terminal amino acid sequence. This will typically, be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include sequences to direct protein trafficking, short peptide sequences which facilitate cloning or purification (e.g. comprising histidine tags i.e. His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences which enhance protein stability. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.

Most preferably, n is 2 or 3.

The Saccharide Antigen

The saccharide antigen is generally the capsular polysaccharide of a GBS or a derivative thereof. Suitable derivatives include oligosaccharide (e.g. from 3 to 150, preferably 8 to 100, monosaccharide units) fragments of the polysaccharide (e.g. refs. 12 to 16), de-acetylated saccharides (Ref. 16), N-acroylated saccharides (16), saccharides with terminal aldehyde groups, etc.

The saccharide is preferably conjugated to a carrier molecule to enhance immunogenicity (e.g. see refs. 4 to 23 etc.). In some embodiments of the invention the GBS saccharide is conjugated to a GBS protein as defined above, thereby giving a polypeptide/saccharide combination of the invention in a single molecule. In other embodiments the GBS saccharide is conjugated to a non-GBS protein, in which case the conjugate will be combined with a separate GBS protein to give a polypeptide/saccharide combination of the invention.

Non-GBS carrier polypeptides include tetanus toxoid, the N. meningitidis outer membrane protein (24), synthetic peptides (25, 26), heat shock proteins (27, 28), pertussis proteins (29, 30), protein D from H. influenzae (31), cytokines (32), lymphokines (32), hormones (32), growth factors (32), toxin A or B from C. difficile (33), iron-uptake proteins (34) etc. Preferred carrier proteins are the CRM197 diphtheria toxoid (35) and tetanus toxoid.

The saccharide and polypeptide are joined covalently. This may involve a direct covalent bond between the saccharide and polypeptide, or indirect coupling via a linker or spacer may be used (e.g. via a B-propionamido linker (16), etc.). Any suitable conjugation chemistry may be used (e.g reductive amination (21) etc.). Linkage is preferably via a terminal saccharide in the polysaccharide.

A single carrier molecule may carry saccharide antigens of a single type (e.g. saccharides derived from a single GBS serotype) or may carry multiple different antigens (e.g. saccharides derived from multiple GBS serotypes, all conjugated to the same carrier).

The saccharides can, of course, be prepared by various means (e.g. purification of the saccharide from GBS, chemical synthesis, etc.), in various sizes (e.g. full-length, fragmented, etc.) and may be derivatised for linking to carriers. They are preferably prepared in substantially pure form (i.e. substantially free from other streptococcal saccharides) or substantially isolated form. Processes for preparing capsular polysaccharides from GBS are well known in the art (e.g. refs. 36 to 39) and processes for preparing oligosaccharides from polysaccharides are also known (e.g. hydrolysis, sonication, enzymatic treatment, treatment with a base followed by nitrosation, etc. (12 to 16)).

As an alternative to using a saccharide antigen in non-conjugated combinations, a peptide mimetic of the GBS capsular polysaccharide may be used (e.g. 40). Suitable peptides can be selected by techniques such as phage display using protective anti-saccharide antibodies. As a further alternative, an anti-idiotypic antibody may be used instead of a saccharide antigen (e.g. ref. 41).

Prime/Boost Schedules

Polypeptide/saccharide combinations of the invention may be given as single doses or as part of a prime/boost schedule. In a prime/boost schedule, the combinations may be used as the priming dose, the boosting dose(s), or both.

If a combination is used for both priming and boosting, it is preferred to use the same combination both times. If a combination is used for only one of priming and boosting, it is preferred that the other dose should use the polypeptide or saccharide on which the combination is based. Thus the invention provides a prime-boost schedule where either (i) one of the saccharide and polypeptide antigens is used for priming an immune response and a combination are used for boosting the response, or (ii) combined saccharide and polypeptide antigens are used for priming an immune response but only one is used for boosting the response.

Various timings for priming and boosting are suitable for use with the invention. In one embodiment, a priming dose is given to a child and a booster is given to a teenager (13-18 years) or young adult (19-25 years). In another embodiment, a priming dose is given to a teenager or young adult and a booster is given during pregnancy. In another embodiment, a priming dose is given to a female who intends to become pregnant and a booster is given during pregnancy.

Immunogenic Pharmaceutical Compositions

Polypeptide/saccharide combinations are formulated as immunogenic compositions, and more preferably as compositions suitable for use as a vaccine in humans (e.g. children or adults). Vaccines of the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat disease after infection), but will typically be prophylactic. Accordingly, the invention includes a method for the therapeutic or prophylactic treatment of GBS infection in an animal susceptible to GBS infection comprising administering to said animal a therapeutic or prophylactic amount of the immunogenic compositions of the invention.

The composition of the invention is preferably sterile.

The composition of the invention is preferably pyrogen-free.

The composition of the invention generally has a pH of between 6.0 and 7.0, more preferably to between 6.3 and 6.9 e.g. 6.6±0.2. The composition is preferably buffered at this pH.

Other components suitable for human administration are disclosed in reference 42.

Vaccines of the invention may be administered in conjunction with other immunoregulatory agents. fu particular, compositions will usually include an adjuvant. Preferred further adjuvants include, but are not limited to, one or more of the following set forth below:

A. Mineral Containing Compositions

Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts. The invention includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc. {e.g. see chapters 8 & 9 of ref. 43}), or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred. The mineral containing compositions may also be formulated as a particle of metal salt. See ref. 44.

B. Oil-Emulsions

Oil-emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59® (5% Squalene, 0.5% TWEEN® 80, and 0.5% SPAN® 85, formulated into submicron particles using a microfluidizer). See ref. 45.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used as adjuvants in the invention.

C. Saponin Formulations

Saponin formulations, may also be used as adjuvants in the invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs.

Saponin compositions have been purified using High Performance Thin Layer Chromatography (HP-LC) and Reversed Phase High Performance Liquid Chromatography (RP-HPLC). Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of production of QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponin formulations may also comprise a sterol, such as cholesterol (see WO 96/33739).

Combinations of saponins and cholesterols can be used to form unique particles called Immunostimulating Complexs (ISCOMs). ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of Quil A, QHA and QHC. ISCOMs are further described in EP 0 109 942, WO 96/11711 and WO 96/33739. Optionally, the ISCOMS may be devoid of additional detergent. See ref. 46.

A review of the development of saponin based adjuvants can be found at ref. 47.

C. Virosomes and Virus Like Particles (VLPs)

Virosomes and Virus Like Particles (VLPs) can also be used as adjuvants in the invention. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins may be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein p1). VLPs are discussed further in WO 03/024480, WO 03/024481, and Refs. 48, 49, 50 and 51. Virosomes are discussed further in, for example, Ref. 52

D. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as:

(1) Non-Toxic Derivatives of Enterobacterial Lipopolysaccharide (LPS)

Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such “small particles” of 3dMPL are small enough to be sterile filtered through a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529. See Ref. 53.

(2) Lipid A Derivatives

Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. OM-174 is described for example in Ref. 54 and 55.

(3) Immunostimulatory Oligonucleotides

Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a sequence containing an unmethylated cytosine followed by guanosine and linked by a phosphate bond). Bacterial double stranded RNA or oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. Optionally, the guanosine may be replaced with an analog such as 2′-deoxy-7-deazaguanosine. See ref. 56, WO 02/26757 and WO 99/62923 for examples of possible analog substitutions. The adjuvant effect of CpG oligonucleotides is further discussed in Refs. 57, 58, WO 98/40100, U.S. Pat. No. 6,207,646, U.S. Pat. No. 6,239,116, and U.S. Pat. No. 6,429,199.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT. See ref. 59. The CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in refs. 60, 61 and WO 01/95935. Preferably, the CpG is a CpG-A ODN. Preferably, the CpG oligonucleotide is constructed so that the 5′ end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3′ ends to form “immunomers”. See, for example, refs. 62, 63, 64 and WO 03/035836.

(4) ADP-Ribosylating Toxins and Detoxified Derivatives Thereof.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (i.e., E. coli heat labile enterotoxin “LT), cholera (“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO 95/17211 and as parenteral adjuvants in WO 98/42375. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G. The use of ADP-ribosylating toxins and detoxified derivaties thereof; particularly LT-K63 and LT-R72, as adjuvants can be found in Refs. 65, 66, 67, 68, 69, 70, 71 and 72 each of which is specifically incorporated by reference herein in their entirety. Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in Domenighini et al., Mol. Microbiol. (1995) 15(6):1165-1167, specifically incorporated herein by reference in its entirety.

E. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor.

F. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in the invention. Suitable bioadhesives include esterified hyaluronic acid microspheres (Ref. 73) or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention. E.g., ref. 74.

G. Microparticles

Microparticles may also be used as adjuvants in the invention. Microparticles (i.e. a particle of 100 nm to 150 pm in diameter, more preferably 200 nm to 30 pm in diameter, and most preferably 500 nm to lO pm in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(a-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide-co-glycolide) are preferred, optionally treated to have a negatively-charged surface (e.g. with SDS) or a positively-charged surface (e.g. with a cationic detergent, such as CTAB).

H. Liposomes

Examples of liposome formulations suitable for use as adjuvants are described in U.S. Pat. No. 6,090,406, U.S. Pat. No. 5,916,588, and EP 0 626 169.

I. Polyoxyethylene ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters. Ref. 75. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with -itn octoxynol (Ref. 76) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (Ref. 77).

Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

J. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in Ref. 78 and 79.

K. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in the invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE).

L. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in the invention include Imiquimod and its homologues, described further in Ref. 80 and 81.

The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention:

-   -   (1) a saponin and an oil-in-water emulsion (ref. 82);     -   (2) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g.,         3dM.PL) (see WO 94/00153);     -   (3) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g.,         3dM.PL)+a cholesterol;     -   (4) a saponin (e.g. QS21)+3dM.PL+IL-12 (optionally+a sterol)         (Ref. 83); combinations of 3d:MPL with, for example, QS21 and/or         oil-in-water emulsions (Ref. 84);     -   (5) SAF, containing 10% squalene, 0.4% TWEEN® 80, 5%         pluronic-block polymer L121, and thr-MDP, either microfluidized         into a submicron emulsion or vortexed to generate a larger         particle size emulsion.     -   (6) RIBI™ adjuvant system (RAS), (Ribi hmnunochem) containing 2%         Squalene, 0.2% TWEEN® 80, and one or more bacterial cell wall         components from the group consisting of monophosphorylipid A         (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS),         preferably M.PL+CWS (DETOX); and     -   (7) one or more mineral salts (such as an aluminum salt)+a         non-toxic derivative of LPS (such as 3dPML).

Aluminium salts and MF59® are preferred adjuvants for parenteral immunisation. Mutant bacterial toxins are preferred mucosal adjuvants.

The composition may include an antibiotic.

GBS polypeptide(s) and saccharide(s) in the compositions of the invention will be present in ‘immunologically effective amounts’ i.e. the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention of disease. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

Typically, the compositions of the invention are prepared as injectables. Direct delivery of the compositions will generally be parenteral (e.g. by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue) or mucosal (e.g. oral or intranasal [85,86]). The compositions can also be administered into a lesion. The invention provides a syringe containing a composition of the invention.

Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated. The vaccines are particularly useful for vaccinating children and teenagers, and more particularly females.

As well as GBS polypeptides and saccharides, the composition of the invention may comprise further antigens. For example, the composition may comprise one or more of the following further antigens:

-   -   antigens from Helicobacter pylori such as CagA [87 to 90], VacA         [91, 92], NAP [93, 94, 95], HopX [e.g. 96], RopY [e.g. 96]         and/or urease.     -   a saccharide antigen from N. meningitidis serogroup A, C, W135         and/or Y, such as the oligosaccharide disclosed in ref. 97 from         serogroup C [see also ref. 98] or the oligosaccharides of ref.         99.     -   a saccharide antigen from Streptococcus pneumoniae [e.g. 100,         101, 102].     -   an antigen from hepatitis A virus, such as inactivated virus         [e.g. 103, 104].     -   an antigen from hepatitis B virus, such as the surface and/or         core antigens [e.g. 104, 105]. an antigen from Bordetella         pertussis, such as pertussis holotoxin (PT) and filamentous         haemagglutinin (FHA) from B. pertussis, optionally also in         combination with pertactin and/or agglutinogens 2 and 3 [e.g.         refs. 106 & 107].     -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter         3 of ref. 108] e.g. the CRM₁₉₇ mutant [e.g. 109].     -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of         ref. 128].     -   a saccharide antigen from Haemophilus influenzae B [e.g. 98].     -   an antigen from hepatitis C virus [e.g. 110].     -   an antigen from N. gonorrhoeae [e.g. 111, 112, 113, 114].     -   an antigen from Chlamydia pneumoniae [e.g. refs. 115 to 121].     -   an antigen from Chlamydia trachomatis [e.g. 122].     -   an antigen from Porphyromonas gingivalis [e.g. 123].     -   polio antigen(s) [e.g. 124, 125] such as OPV or, preferably,         IPV.         -   rabies antigen(s) [e.g. 126] such as lyophilised inactivated             virus [e.g. 127, RABAVERT™], measles, mumps and/or rubella             antigens [e.g. chapters 9, 10 & 11 of ref. 128].     -   influenza antigen(s) [e.g. chapter 19 of re£ 128], such as the         haemagglutinin and/or neuraminidase surface proteins.     -   an antigen from Moraxella catarrhalis [e.g. 129].     -   an antigen from Streptococcus pyogenes (group A streptococcus)         [e.g. 3, 130, 131].     -   an antigen from Staphylococcus aureus [e.g. 132].     -   an antigen from Bacillus anthracis [e.g. 133, 134, 135].     -   an antigen from a virus in the flaviviridae family (genus         flavivirus), such as from yellow fever virus, Japanese         encephalitis virus, four serotypes of Dengue viruses, tick-borne         encephalitis virus, West Nile virus.     -   a pestivirus antigen, such as from classical porcine fever         virus, bovine viral diarrhoea virus, and/or border disease         virus.     -   a parvovirus antigen e.g. from parvovirus B19.     -   a prion protein (e.g. the CJD prion protein)     -   an amyloid protein, such as a beta peptide [136]     -   a cancer antigen, such as those listed in Table 1 of ref. 137 or         in tables 3 & 4 of ref. 138.

The composition may comprise one or more of these further antigens.

Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means [107]).

Where a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens. DTP combinations are thus preferred. Saccharide antigens are preferably in the form of conjugates. Carrier proteins for the conjugates are the same as those described above for GBS saccharide conjugation, with CRM197 being preferred.

Antigens in the composition will typically be present at a concentration of at least 1 μg/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.

As an alternative to using protein antigens in the composition of the invention, nucleic acid encoding the antigen may be used. Protein components of the compositions of the invention may thus be replaced by nucleic acid (preferably DNA e.g. in the form of a plasmid) that encodes the protein.

Methods of Treating Patients

The invention provides polypeptide/saccharide combinations of the invention for use as medicaments. The medicament is preferably able to raise an immune response in a mammal (i.e. it is an immunogenic composition) and is more preferably a vaccine.

The invention also provides a method of raising an immune response in a patient, comprising administering to a patient a composition of the invention. The immune response is preferably protective against streptococcal disease, and may comprise a humoral immune response and/or a cellular immune response.

The invention also provides the use of polypeptide/saccharide combination of the invention in the manufacture of a medicament for raising an immune response in an patient. The medicament is preferably an immunogenic composition (e.g. a vaccine). The medicament is preferably for the prevention and/or treatment of a disease caused by GBS (e.g. meningitis, sepsis, chorioamnionitis).

The invention also provides for a kit comprising a first component comprising the immunogenic compositions of the invention. The kit may further include a second component comprising one or more of the following: instructions, syringe or other delivery device, adjuvant, or pharmaceutically acceptable formulating solution.

The invention also provides a delivery device pre-filled with the immunogenic compositions of the invention.

The invention also provides a method for raising an immune response in a mammal comprising the step of administering an effective amount of a composition of the invention. The immune response is preferably protective and preferably involves antibodies and/or cell-mediated immunity. The method may raise a booster response.

Process for Manufacturing

The invention provides a process for preparing a composition of the invention, comprising the step of mixing (i) one or more GBS polypeptide antigens with (ii) one or more GBS saccharide antigens.

The process may comprise the step of covalently linking the GBS polypeptide to the GBS saccharide in order to form a conjugate.

DEFINITIONS

The term “comprising” means “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example, x±10%.

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

MODES FOR CARRYING OUT THE INVENTION

GBS serotype III is grown in Todd-Hewitt broth as described in reference 36 and its capsular polysaccharide was purified. The polysaccharide is depolymerised, sized and purified as described in reference 14 to give oligosaccharide antigen. Similar procedures are used to prepare capsular polysaccharides from other GBS serotypes.

The oligosaccharide is either admixed with or covalently conjugated (directly or via a linker) to purified serotype V protein. Preferably, the protein comprises a GBS antigen or a fragment thereof selected from the group consisting of GBS 80, GBS 91, GBS 104, GBS 147, GBS 173, GBS 276, GBS 305, GBS 313, GBS 322, GBS 328, GBS 330, GBS 338, GBS 358, GBS 361, GBS 404, GBS 656, GBS 690, and GBS 691.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. All documents cited herein are incorporated by reference in their entirety.

REFERENCES The Contents of which are Hereby Incorporated by Reference

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1. An immunogenic composition comprising: (a) an isolated Group B streptococcus (GBS) saccharide antigen selected from GBS serotype Ia, Ib, and III, and (b) a combination comprising two isolated GBS polypeptide antigens, wherein a first isolated GBS polypeptide antigen is selected from GBS 80 set forth as SEQ ID NO:2 and immunogenic fragments of the GBS 80 comprising at least 7 consecutive amino acids of SEQ ID NO:2; and wherein a second GBS polypeptide antigen is selected from GBS 328 set forth as SEQ ID NO:20 and immunogenic fragments of the GBS 328 comprising at least 7 consecutive amino acids of SEQ ID NO:20.
 2. The immunogenic composition of claim 1, wherein the combination consists of the two isolated GBS polypeptide antigens.
 3. The immunogenic composition of claim 1, wherein the first isolated GBS polypeptide antigen is GBS 80 set forth as SEQ ID NO:2.
 4. The immunogenic composition of claim 1, wherein the second isolated GBS polypeptide is GBS 328 set forth as SEQ ID NO:20.
 5. The immunogenic composition of claim 4, wherein the first isolated GBS polypeptide antigen is GBS80 as set forth in SEQ ID NO:2.
 6. The immunogenic composition of claim 1, wherein said composition further comprises a third isolated GBS polypeptide antigen.
 7. The immunogenic composition of claim 6, which comprises the GBS 80 set forth as SEQ ID NO:2 and GBS691 set forth as SEQ ID NO:36.
 8. The immunogenic composition of claim 1 wherein the isolated GBS saccharide antigen is a GBS serotype Ia saccharide antigen.
 9. The immunogenic composition of claim 1 further comprising a diphtheria toxoid.
 10. The immunogenic composition of claim 9 wherein the diphtheria toxoid is CRM197.
 11. The immunogenic composition of claim 1, wherein at least one of the isolated GBS polypeptide antigens is covalently linked to the GBS saccharide antigen.
 12. The immunogenic composition of claim 1, wherein said GBS saccharide antigen is covalently linked to a carrier protein.
 13. The immunogenic composition of claim 12, wherein said carrier protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid, N. meningitidis outer membrane protein, heat shock protein, pertussis protein, protein D from H. influenzae, and toxin A or B from C. difficile.
 14. The immunogenic composition of claim 12, wherein said carrier protein is selected from the group consisting of tetanus toxoid and diphtheria toxoid.
 15. The immunogenic composition of claim 14, wherein said carrier protein is a diphtheria toxoid.
 16. The immunogenic composition of claim 15, wherein said diphtheria toxoid is CRM197.
 17. A method for the therapeutic or prophylactic treatment of GBS infection in an animal susceptible to GBS infection comprising administering to said animal a therapeutic or prophylactic amount of the immunogenic composition of claim
 1. 18. A method for the manufacture of a medicament for raising an immune response against GBS comprising combining: an isolated Group B streptococcus (GBS) saccharide antigen selected from GBS serotype Ia, Ib, and III; and at least two isolated GBS polypeptide antigens, wherein the wherein a first isolated GBS polypeptide antigen is selected from GBS 80 set forth as SEQ ID NO:2 and immunogenic fragments of the GBS 80 comprising at least 7 consecutive amino acids of SEQ ID NO:2; and wherein a second GBS polypeptide antigen is selected from GBS 328 set forth as SEQ ID NO:20 and immunogenic fragments of the GBS 328 comprising at least 7 consecutive amino acids of SEQ ID NO:20. 